Examination of the pregnancy-associated plasma protein-A assay on the Beckman Coulter Access® platform: suitability for use in first trimester Down's syndrome screening

Abstract

Objective

To explore the clinical validity of the new Access® pregnancy-associated plasma protein-A (PAPP-A) assay for Down's syndrome screening.

Setting

Academic hospital.

Method

Residual serum samples (n = 416) received for routine first trimester Down's syndrome screening (10-13 weeks) were retrieved from freezer storage and tested using two PAPP-A immunoassay methods. The new Access® assay was specifically compared, on a subset of these samples (n = 194), with an assay proven to give acceptable Down's syndrome screening performance, the PerkinElmer (PE) AutoDELFIA method. Access® PAPP-A levels were examined in relation to gestational age and maternal weight, and were compared with the PE method by regression and Bland-Altman analyses.

Results

Access® PAPP-A assay results were highly correlated with the PE AutoDELFIA method (r = 0.97). PAPP-A levels obtained using the Access® assay increased with advancing gestational age and were inversely related to maternal weight, as expected. The distribution of multiples of the median (MoM) values fit a log Gaussian distribution and the standard deviation of the log MoM (0.2331) matched published estimates. PAPP-A MoM levels in Down's syndrome pregnancies (n = 6, median 0.30) showed the expected reduction.

Conclusion

Using an appropriate gestational age-specific median equation, the Access® PAPP-A assay is expected to perform acceptably in first trimester Down's syndrome screening.

INTRODUCTION

Screening for Down's syndrome risk in the first trimester of pregnancy involves serum measurement of free beta human chorionic gonadotropin and pregnancy-associated plasma protein-A (PAPP-A), along with ultrasound measurement of nuchal translucency.^{1, 2} Large multicentre trials in the USA³ and Europe⁴ have shown this combined testing to achieve an 85% Down's syndrome detection rate with an associated 5-6% false-positive rate.

One of the barriers to implementing the combined test in the USA has been assay availability. PAPP-A immunoassay methods are in limited supply and no method has yet received clearance from the US Food and Drug Administration. One widely used method in the USA (Beckman Coulter Inc, Chaska, MN, USA, originally manufactured by Diagnostic Systems Labs Inc, Webster, TX, USA) is a manual enzyme-linked immunosorbent assay that is labour intensive and time consuming. The goal of this study was to evaluate a new automated method for PAPP-A measurement using the Beckman Coulter Access® platform. The analytical performance of this assay has been previously described.⁵ We aimed to confirm and extend these findings by comparing Access® results with a method with widely proven Down's syndrome screening performance (PerkinElmer AutoDELFIA, Waltham, MA, USA). Specifically, we examined first trimester median PAPP-A levels using the Access® assay, the maternal weight effect, the distribution of PAPP-A values obtained with the Access® assay, and documented the well-described reduction in first trimester PAPP-A levels in a small group of women with Down's syndrome pregnancies.

Methods

Residual serum samples were available as part of routine first trimester prenatal screening performed by the Division of Medical Screening and Special Testing at Women and Infants Hospital. Serum samples that had been collected between May and November 2007 were kept in freezer storage at -20°C. Samples from singleton pregnancies occurring in Caucasian women were eligible. Those reporting in vitro fertilization, or those with a known chromosomal abnormality were excluded. Approximately equal numbers of samples satisfying these criteria were randomly chosen within each week, except for 10 weeks, where fewer samples were available. A total of six samples from pregnancies affected by Down's syndrome were also available in the same gestational age range, but from a different time period (August 2007 through May 2009). This study was conducted with the approval of the Institutional Review Board for Human Studies at Women and Infants Hospital. A total of 416 samples were selected that covered the gestational age range of 10-13 completed weeks, as determined by crown rump length ultrasound measurements. Samples were retrieved from freezer storage and tested for PAPP-A using the automated Beckman Coulter (Access®) method. A subset of these samples (n = 194), covering the full range of results, were also run on the PerkinElmer AutoDELFIA (PE).

Method comparisons were performed by least-squares linear regression and Bland-Altman analyses⁶ after appropriate logarithmic transformations. In order to compare the existing PAPP-A method, the values of which are expressed in mlU/mL, with those reported by Access® in ng/mL, a conversion factor specific to these kit lots/samples was calculated and applied. Access® PAPP-A results were regressed versus gestational age (in days) to generate medians. These were used to convert all results into multiple of the median (MoM) levels that were then weight-adjusted using a published reciprocal model.⁷ The PAPP-A levels in the six Down's syndrome affected pregnancies were also converted to weight-adjusted MoM levels using the same medians and weight adjustment factors. The standard deviation (SD) for the first trimester PAPP-A weight-adjusted MoM levels (after a logarithmic transformation) was derived by computing the slope of the line fitted to the data in a probability plot over the range of values that appeared linear. This SD was then compared with others estimated in the literature for first trimester PAPP-A measurements. Tests of significance were two-sided, at a P ≤ 0.05 level. Graphics and select analyses were created using PRISM software (GraphPad, La Jolla, CA, USA). Additional analyses were performed using Excel (Microsoft, Redmond, OR, USA) or BMPD (Statistical Solutions, Saugus, MA, USA).

RESULTS

Method comparison

PAPP-A levels were compared between PE and Access® platforms. Since the PE assay is calibrated in mlU/mL while the Access® assay is calibrated in ng/mL, we created a ‘conversion factor’ so that comparisons could be on a common ng/mL scale. On average, 1 IU/mL was equivalent to 411 ng/mL (e.g. PE PAPP-A in mlU/mL × 411 = Access® PAPP-A in ng/mL). This conversion factor (411 ng/IU) was reasonably constant over the range of PAPP-A values. Figure 1a shows the scatterplot (on logarithmic scales) for the paired results. The correlation is 0.97 and the data appear close to the dashed line of equality. The Bland-Altman analysis (Figure 1b) shows a small but significant negative slope shown by the solid line (ratio = -0.0734 × average PAPP-A + 0.2155, P < 0.001). On average, the Access®/PE ratio is 1.02 (Access® is 2% higher than PE) with the 95% prediction limits⁸ of 0.75-1.40 indicated by dotted lines.

Figure 1

Bland-Altman method comparison for pregnancy-associated plasma protein-A (PAPP-A). Paired PAPP-A results from unaffected pregnancies (open circles) are shown on a logarithmic scale (a, in the left-hand column) in the form of a scatterplot. PerkinElmer AutoDELFA (PE) results are adjusted to ng/mL for comparison purposes, as described in the text. The dashed line of equality (Y = X) is provided. The corresponding Bland-Altman analyses are provided in the right-hand column (b). The horizontal axis is the geometric mean of the paired PAPP-A results while the vertical axis shows the ratio of the two values on a logarithmic scale. The dashed line is drawn at a ratio of 1.0 (when both assays provide identical results). The solid line represents the best fit line through the data, with the dotted lines showing the corresponding 95% prediction limits. Numbers of samples, correlation, slopes and tests of significance are provided in the text.

Medians and distribution of data

Median levels of PAPP-A by gestational age were calculated for the Access® assay (Table 1) using a log-linear regression analysis of value in ng/mL against gestational age in days (median PAPP-A value = 10^{(2.3109+(days-60)x0.02306)}). Weighting was by the square root of the number of observations at each week. Assay results in ng/mL were then converted to MoM by dividing the day-specific median. The expected inverse relationship between PAPP-A MoM and maternal weight was observed (Figure 2). The PAPP-A MoM levels were then adjusted for maternal weight using a weighted reciprocal equation, fitted to the average maternal weight in pounds and associated median PAPP-A MoM levels, that best described the association (expected MoM= (202.2508 × 1/weight) - 0.32681). The maternal weight adjustment included truncation limits of 100 and 250 pounds.

Table 1

First trimester PAPP-A median levels using the Access^® platform

Completed week	Average GA (days)	Number of observations	Median PAPP-A (ng/mL)	Regressed^* median (ng/mL)
10	73.2	74	412	412
11	80.7	121	616	614
12	87.1	110	861	863
13	93.3	111	1413	1199

PAPP-A, pregnancy-associated plasma protein-A; GA, gestational age;

Median = 10^{(2.3109+(days-60)×0.02306}

Figure 2

Maternal weight effect for pregnancy-associated plasma protein-A (PAPP-A) measurements in the first trimester using the Access⁸ assay. Maternal weight and corresponding PÀPP-A measurements for 416 first trimester maternal serum samples (open circles), along with the observed medians (open squares) and regression line (expected PAPP-A MoM = 202.2508 × 1/maternal_weight_in_pounds - 0.32681). MoM, multiple of the median

The distribution of PAPP-A MoM levels was compared with published distribution parameters as a means to verify reliable Down's syndrome risk assignment using the Access® assay method. Figure 3 shows the weight-adjusted PAPP-A MoM levels from the Access® platform on a probability plot, after a logarithmic transformation. A straight line indicates the data fit a log Gaussian distribution well. The slope of the line fitted to the data on that plot over the entire range of values provided a reliable estimate of the SD. The computed logarithmic SD is 0.2331. Table 2 contains published estimates of the PAPP-A log SD in unaffected pregnancies along with the estimate for the Access® platform derived in the present study.^2,4,9–19 The Access® estimate is well within the published range, close to an estimate widely used when calculating Down's syndrome risk.⁴

Table 2

Comparison of our study's estimate for the PAPP-A MoM standard deviation in unaffected pregnancies, compared with other published estimates

			Unaffected pregnancies
Publication	Assay method	Gestational age (weeks)	Number	Standard deviation^*
Bersinger et al.⁹	In-house^†	9-14	31	0.2054
Brambati et al.¹⁰	RIA	9-12	89	0.2217
Canick et al.¹¹	DSL ELISA	10-13	33,459	0.2260
Current study	Access^®	10-13	416	0.2331
Bindra et al.¹²	Kryptor	9-14	14,607	0.2361
Wald et al.⁴	AutoDELFIA	10-13	420	0.2495
De Biasio et al.¹³	Immunoradiometric^‡	10-13	1454	0.2500
Muller et al.¹⁴	RIA^¶	9-14	66	0.2530
Wald et al.¹⁵	AutoDELFIA	9-13	383	0.2659
Haddow et al.²	In-house^†	9-14	265	0.2880
Van Lith¹⁶	RIA	9-12	280	0.2975
Qin et al.¹⁷	In-house^†	7-12	306	0.3359
Bersinger et al.¹⁸	In-house^†	10-13	210	0.3729

ELISA, enzyme-linked immunosorbent assay; PAPP-A, pregnancy-associated plasma protein-A; MoM, multiple of the median

Calculated after a logarithmic transformation of the PAPP-A MoM results

†

Antibodies from DAKO in ELISA or immunofluorometric formats

‡

Ortho Clinicals Diagnostic, Amersham, UK; no longer available

RIA = radioimmunoassay¹⁹

Figure 3

Probability plot for first trimester pregnancy-associated plasma protein-A (PAPP-A) using the Access assay. MoM levels on a log scale of PAPP-A values. The Down's syndrome samples tested are shown in closed squares. MoM, multiple of the median

To examine Access® PAPP-A results in first trimester maternal serum from Down's syndrome pregnancies directly, residual serum was tested from six confirmed cases. The median level for the Access® assay was 0.30 MoM (range 0.10-1.50 MoM) in these samples. The median level reported for PAPP-A in Down's syndrome pregnancy ranges from 0.34 to 0.58 MoM.⁴ These six data points (filled squares) have been included in Figure 3 for comparison with the levels found in unaffected pregnancies.

DISCUSSION

We have examined the Beckman Coulter Access® PAPP-A assay for use in the late first trimester of pregnancy. The Access® assay shows a high correlation with the Perkin Elmer PAPP-A assay which has proven to perform well in first trimester Down's syndrome screening.⁴ Furthermore, the Access® results show the expected relationship with maternal weight, and PAPP-A MoM levels in first trimester unaffected pregnancies have an appropriate distribution, as shown by the probability plot and the SD of the log MoM. The current estimate of 0.2331 as the log SD is one of the reported estimates showing the lowest population variability. The low PAPP-A MoM level found in first trimester samples from women with Down's syndrome pregnancies provides further evidence for the suitability of this new platform. A separate study⁵ concluded that the Access® assay for PAPP-A demonstrated appropriate analytical performance and results agreed with another assay proven effective in Down's syndrome screening,³ the manual PAPP-A assay from Beckman Coulter Inc.

One other automated method for PAPP-A measurement, on the Immulite 2000 platform (Siemens Healthcare Diagnostics, Deerfield, IL, USA) has been available to some extent in the USA. Only one study has reported its performance in screening; the Immulite PAPP-A method was compared with the Brahms Kryptor platform (Annapolis, MD, USA, PAPP-A reagents not available in the USA) in 2005.²⁰ The Immulite was reported to have a high bias in cases leading to a PAPP-A median MoM of 0.62 in Down's syndrome, versus a median of 0.47 MoM using the Kryptor, suggesting a relative insensitivity for the Immulite method. Further study of the Immulite PAPP-A assay performance for Down's syndrome screening is required. In other studies, the Kryptor and PE assays were shown to give comparable results.²¹

The absence of an international reference standard is a continuing issue in striving to improve uniformity of PAPP-A results between laboratories and methods. The Access® instrument calibrates PAPP-A levels in ng/mL²² versus other methods that use mlU/mL as the reporting unit. Although disconcerting, this underscores the need for each prenatal screening laboratory to carefully monitor its median MoM levels over time regardless of the methodology chosen, and revise medians when necessary. A shared calibration material for PAPP-A assays would allow for more rigorous method comparisons and perhaps improve performance.

One strategy to understand the degree of agreement between laboratories that use PAPP-A measurement is inter-laboratory proficiency programmes. One such programme exists in Europe (UK NEQAS, www.ukneqas.org.uk). Our group at Women and Infants Hospital (Providence, RI, USA) have recently introduced a similar Inter-laboratory Comparison Program for the USA and elsewhere (ICP, www.ipmms.org).²³ In the 2009 FT-C Survey, PAPP-A levels were reported by 26 participants using four different assay methods (Access®, DSL, PE, and Siemens Immulite). Among the five sample challenges, the coefficients of variation (CVs) for the 14 participants reporting in IU/mL and the 12 reporting in ng/mL were all 11% or lower, except for one sample with a very low target PAPP-A value. The corresponding MoM levels for the 24 clinical laboratories were combined into one analysis, with CVs for the five challenges ranging between 22% and 28%. These data suggest that continuing efforts need to be directed towards improving PAPP-A assay correspondence or agreement across platforms.

In summary, the Access® assay provides a simple and reliable method for the measurement of PAPP-A in pregnancy serum and is expected to give appropriate performance as a first trimester marker of Down's syndrome. Further progress in calibration and availability of PAPP-A assays, especially in the USA, would be helpful to laboratories interested in its clinical use.

Footnotes

Acknowledgment

This study was funded in part by Beckman Coulter Inc.

References

Wald

, Hackshaw

. Combining ultrasound and biochemistry in first-trimester screening for Down's syndrome. Prenat Diagn 1997; 17: 821–9

Haddow

, Palomaki

, Knight

. Screening of maternal serum for fetal Down's syndrome in the first trimester. N Engl J Med 1998; 338: 955–61

Malone

, Canick

, Ball

. First-trimester or second-trimester screening, or both, for Down's syndrome. N Engl J Med 2005; 353: 2001–11

Wald

, Rodeck

, Hackshaw

. First and second trimester antenatal screening for Down's syndrome: the results of the Serum, Urine and Ultrasound Screening Study (SURUSS). J Med Screen 2003; 10: 56–104

Owen

, Rawlins

, La'ulu

. Performance characteristics of the Access pregnancy-associated plasma protein-A assay. Clin Chim Acta 2008; 398: 165–7

Bland

, Altman

. Measuring agreement in method comparison studies. Stat Methods Med Res 1999; 8: 135–60

Neveux

, Palomaki

, Larrivee

. Refinements in managing maternal weight adjustment for interpreting prenatal screening results. Prenat Diagn 1996; 16: 1115–19

Twomey

. How to use difference plots in quantitative method comparison studies. Ann Clin Biochem 2006; 43: 124–9

Bersinger

, Marguerat

, Pescia

. Pregnancy-associated plasma protein A (PAPP-A): measurement by highly sensitive and specific enzyme immunoassay, importance of first-trimester serum determinations, and stability studies. Reprod Fertil Dev 1995; 7: 1419–23

10.

Brambati

, Tului

, Bonacchi

. Serum PAPP-A and free beta-hCG are first-trimester screening markers for Down syndrome. Prenat Diagn 1994; 14: 1043–7

11.

Canick

, Lambert-Messerlian

, Palomaki

. Comparison of serum markers in first-trimester Down syndrome screening. Obstet Gynecol 2006; 108: 1192–9

12.

Bindra

, Heath

, Liao

. One-stop clinic for assessment of risk for trisomy 21 at 11-14 weeks: a prospective study of 15 030 pregnancies. Ultrasound Obstet Gynecol 2002; 20: 219–25

13.

De Biasio

, Siccardi

, Volpe

. First-trimester screening for Down syndrome using nuchal translucency measurement with free beta-hCG and PAPP-A between 10 and 13 weeks of pregnancy - the combined test. Prenat Diagn 1999; 19: 360–3

14.

Muller

, Cuckle

, Teisner

. Serum PAPP-A levels are depressed in women with fetal Down syndrome in early pregnancy. Prenat Diagn 1993; 13: 633–6

15.

Wald

, George

, Smith

. Serum screening for Down's syndrome between 8 and 14 weeks of pregnancy. International Prenatal Screening Research Group. Br J Obstet Gynaecol 1996; 103: 407–12

16.

Van Lith

, Grudzinskas

, Mantingh

, Shrimanker

, Suzuki

, Macintosh

MCM

. First trimester maternal serum PAPP-A levels in pregnancies with chromosomally normal and abnormal fetuses. published doctoral thesis, Rijksuniversiteit Groningen, The Netherlands, 1994 (ISBN 90-9007280-2)

17.

Qin

, Nguyen

, Christiansen

. Time-resolved immunofluorometric assay of pregnancy-associated plasma protein A in maternal serum screening for Down's syndrome in first trimester of pregnancy. Clin Chim Acta 1996; 254: 113–29

18.

Bersinger

, Brizot

, Johnson

. First trimester maternal serum pregnancy-associated plasma protein A and pregnancy-specific beta 1-glycoprotein in fetal trisomies. Br J Obstet Gynaecol 1994; 101: 970–4

19.

Sinosich

, Teisner

, Folkersen

. Radioimmunoassay for pregnancy-associated plasma protein A. Clin Chem 1982; 28: 50–3

20.

Spencer

. First trimester maternal serum screening for Down's syndrome: an evaluation of the DPC Immulite 2000 free beta-hCG and pregnancy-associated plasma protein-A assays. Ann Clin Biochem 2005; 42: 30–40

21.

Wojdemann

, Larsen

, Rode

. First trimester Down syndrome screening: distribution of markers and comparison of assays for quantification of pregnancy-associated plasma protein-A. ScandJ Clin Lab Invest 2006; 66: 101–11

22.

Bohm

, Grudzinskas

, Rosen

. Reference preparation for assay of some pregnancy and cancer associated proteins. Lancet 1980; 2: 796

23.

Palomaki

, Knight

, Lambert-Messerlian

, Canick

, Haddow

. Four years experience with an Inter-Laboratory Comparison Program involving first trimester markers for Down syndrome. Arch Pathol Lab Med 2010 (in press)