Reply of the authors:

Abstract

We thank Dr. Ferrero et al. for their comment on our recent article (1Mueller M.D. Raio L. Spoerri S. Ghezzi F. Dreher E. Bersinger N.A. Novel placental and nonplacental serum markers in ectopic versus normal intrauterine pregnancy.Fertil Steril. 2004; 81: 1106-1111Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We were aware that the levels of vascular endothelial growth factor (VEGF) determined in peripheral blood would depend on the platelet count and, practically, on the use of anticoagulants. Ferrero et al. correctly stated that standardization of blood sampling could be clinically unsuitable; indeed, individual information on clotting time was not available retrospectively, and platelet count was not performed. Pregnancy-associated plasma protein A (PAPP-A) binds strongly to heparin (2Sinosich M.J. Zakher A. Pregnancy-associated plasma protein A interaction with heparin: a critical appraisal.Gynecol Obstet Invest. 1991; 32: 72-77Crossref PubMed Scopus (3) Google Scholar), and interaction with other anticoagulants was suggested more than 2 decades ago (3Toop K.M. Klopper A. Effect of anticoagulants on the measurement of pregnancy-associated plasma protein A (PAPP-A).Br J Obstet Gynaecol. 1983; 90: 150-155Crossref PubMed Scopus (11) Google Scholar). For these reasons, it has long become the accepted standard to use serum only for the determination of PAPP-A, which has gained importance in antenatal care (trisomy 21 screening). Very recently, the mandatory use of serum has been demonstrated also for the automated immunoassay system most frequently used for PAPP-A determination (4Spencer K. The influence of different sample collection types on the levels of markers used for Down's syndrome screening as measured by the Kryptor immunoassay system.Ann Clin Biochem. 2003; 40: 166-168Crossref PubMed Scopus (24) Google Scholar). The control samplesin our study (intrauterine pregnancies) were serum only; they were extracted from the first-trimester chromosomal abnormality screening program, as stated in our article. Moreover, in the current clinical context of extrauterine pregnancy (emergency referrals), it was not possible to collect different tubes of peripheral blood. We can assume that no bias regarding clotting time was introduced between cases and controls. Centrifugation was always at 1800 × g for 10 minutes in a cooled Beckman centrifuge. We had previously used without problems the Duo-Set assay method (R&D Systems Europe, Oxford, United Kingdom) for the determination of VEGF in culture supernatants and in biological fluids other than serum, but we found that Duo-Set yielded occasional high VEGF values in serum. We contacted the manufacturer, who suggested that we use the Quantikine-VEGF ELISA kit; this was introduced successfully — the same sera did not produce high VEGF results with the new protocol. R&D Systems has since removed the recommendation for use with serum from the Duo-Set instructions. The instructions in the Quantikine protocol recommend that this test to be used with either serum or plasma and give a range for serum of 62–707 pg/mL VEGF, whereas the antigen is quoted as nondetectable in citrate plasma; information on pregnancy samples is not given. In our study, the VEGF range in the controls was considerably less, between <5 and <300 pg/mL, with a median of >10 pg/mL. The suggested triple marker analysis thus might or might not be improved with the use of citrate plasma for VEGF determination; PAPP-A, however, should always be assayed in serum. “Triple marker” for ectopic pregnancyFertility and SterilityVol. 82Issue 4PreviewMueller et al. (1) should be congratulated for their proposal of a “triple marker analysis” [vascular endothelial growth factor (VEGF)/(pregnancy-associated plasma protein A × P)] in the diagnosis of ectopic pregnancy. We would like to report some observations on blood sampling and processing that might improve the value of this marker combination. Full-Text PDF