Evaluation of a dried blood spot assay to measure prenatal screening markers pregnancy-associated plasma protein a and free ß-subunit of human chorionic gonadotropin.

BACKGROUND: First-trimester prenatal screening for aneuploidy by use of dried blood spots (DBSs) may offer practical benefits in settings where the instability of intact human chorionic gonadotropin (hCG) is problematic. We evaluated a DBS pregnancy-associated plasma protein A (PAPP-A) and free ß-subunit of hCG (free hCGß) dual assay and compared it to serum screening. METHODS: Hematocrit-corrected DBS PAPP-A and free-hCGß concentrations were measured and compared with serum concentrations in 252 first-trimester samples. Serum intact hCG was also measured and, with serum free hCGß, was used to fit a model to predict serum-equivalent DBS free-hCGß concentrations. In a separate experiment, we investigated the effects of temperature and relative humidity during the blood spot drying process. RESULTS: The DBS assay for PAPP-A performed similarly to the serum assay, whereas free-hCGß DBS measurements were consistently higher than in serum. Purifying blood spots of intact hCG suggested that the free-hCGß DBS assay is measuring a composite of free hCGß and additional ß-subunits from intact hCG. The drying experiment showed that increased temperature and relative humidity during the drying process resulted in increased free hCGß and reduced PAPP-A. CONCLUSIONS: Despite measuring additional free hCGß compared to the serum assay, DBS analysis has a role in first-trimester combined screening for trisomy 21.

The stability of free-ß human chorionic gonadotrophin and pregnancy-associated plasma protein-A in first trimester dried blood spots.

OBJECTIVES: To determine the stability of first trimester free-ß human chorionic gonadotrophin (free-ß hCG) and pregnancy-associated plasma protein-A (PAPP-A) in dried blood spots (DBSs) under typical storage conditions. METHODS: First trimester maternal blood was spotted onto filter paper and left to dry. DBSs were analysed for PAPP-A and free-ß hCG using an AutoDELFIA dual assay at t = 0. Cards were stored at one of - 20 °C, refrigerator temperature, room temperature or 30 °C and reanalysed at set future time points. RESULTS: Free-ß hCG was stable (<10% change in concentration) under all temperatures tested for at least 35 days. PAPP-A was stable at - 20 °C and refrigerator temperature for at least 35 days. However, PAPP-A levels decreased by 10% at 4.1 days at room temperature and at 3.9 days at 30 °C. Longer-term storage at - 20 °C and refrigerator temperature showed that both PAPP-A and free-ß hCG levels were significantly decreased by 107 and 244 days. CONCLUSIONS: Free-ß hCG stability is greatly improved in DBS compared to serum storage; however PAPP-A stability is decreased in the DBS medium. Despite this DBS, screening may not necessitate such strict storage and transportation rules compared to serum screening programmes.

A dissociative fluorescence enhancement technique for one-step time-resolved immunoassays.

The limitation of current dissociative fluorescence enhancement techniques is that the lanthanide chelate structures used as molecular probes are not stable enough in one-step assays with high concentrations of complexones or metal ions in the reaction mixture since these substances interfere with lanthanide chelate conjugated to the detector molecule. Lanthanide chelates of diethylenetriaminepentaacetic acid (DTPA) are extremely stable, and we used EuDTPA derivatives conjugated to antibodies as tracers in one-step immunoassays containing high concentrations of complexones or metal ions. Enhancement solutions based on different ß-diketones were developed and tested for their fluorescence-enhancing capability in immunoassays with EuDTPA-labelled antibodies. Characteristics tested were fluorescence intensity, analytical sensitivity, kinetics of complex formation and signal stability. Formation of fluorescent complexes is fast (5 min) in the presented enhancement solution with EuDTPA probes withstanding strong complexones (ethylenediaminetetra acetate (EDTA) up to 100 mM) or metal ions (up to 200 µM) in the reaction mixture, the signal is intensive, stable for 4 h and the analytical sensitivity with Eu is 40 fmol/L, Tb 130 fmol/L, Sm 2.1 pmol/L and Dy 8.5 pmol/L. With the improved fluorescence enhancement technique, EDTA and citrate plasma samples as well as samples containing relatively high concentrations of metal ions can be analysed using a one-step immunoassay format also at elevated temperatures. It facilitates four-plexing, is based on one chelate structure for detector molecule labelling and is suitable for immunoassays due to the wide dynamic range and the analytical sensitivity.

First trimester Down's syndrome screening shows high detection rate for trisomy 21, but poor performance in structural abnormalities--regional outcome results.

OBJECTIVE: To evaluate whether first trimester screening markers are altered in pregnancies affected both by other chromosomal defects than trisomy 21 and structural anomalies and whether it is possible to detect these pregnancies by combined ultrasound and biochemical screening test. METHODS: Altogether 4,776 singleton pregnancies underwent first trimester screening. Of them, 3,101 women were screened using a combination of maternal serum free hCG, pregnancy-associated plasma protein A and nuchal translucency and 1,361 women with first trimester biochemistry without ultrasound. Nuchal translucency screening was performed between the 11th and 13+6th gestational weeks, and biochemistry 1-2 weeks earlier. RESULTS: Using a fixed cut-off rate of 1:250 for Down's syndrome, the detection rate of trisomies 21, 18 and 13 were 92, 67 and 0%, respectively. All open defects, 85% of cardiac defects and other minor defects were not detected in first trimester screening. Majority of these structural abnormalities occurred in women under 35 years of age. CONCLUSION: First trimester Down's syndrome screening is effective in trisomy screening, but its performance in structural abnormalities is low, when used as a part of routine clinical practice. We conclude that it is too early to drop second trimester screening ultrasound entirely from antenatal care programs if a high detection rate is to be achieved also in structural defects.

Maternal serum human chorionic gonadotrophin and pregnancy-associated plasma protein A in twin pregnancies in the first trimester.

OBJECTIVES: To determine the levels of free beta-human chorionic gonadotrophin (beta-hCG) and pregnancy-associated plasma protein A (PAPP-A) in twin pregnancies in the first trimester. METHODS: Serum samples were obtained from 67 pregnant women with twin pregnancies and maternal serum free beta-hCG and PAPP-A concentrations were compared with those of 4279 singleton controls between the 8th and 13th weeks of gestation. RESULTS: The geometric means of chromosomally normal twin pregnancies were 1.85 MoM for free beta-hCG and 2.36 MoM for PAPP-A. There were no cases affected by Down syndrome in either group. CONCLUSION: Twin pregnancies secrete more PAPP-A than expected on the basis of singleton controls whereas free beta-hCG production is not increased. The results of the present study can be used to establish normal reference values when introducing first trimester Down syndrome screening in prenatal care.