Replicate real-time PCR testing of DNA in maternal plasma increases the sensitivity of non-invasive fetal sex determination.

BACKGROUND: We determined fetal sex in pregnancies referred for invasive prenatal diagnosis procedures by analysis of DNA in maternal plasma. METHODS: Twelve pregnancies at risk of X-linked haemophilia and 32 pregnancies at risk of chromosomal aneuploidies at a gestational age ranging from 10 to 18 weeks recruited before chorionic villus sampling or amniocentesis were involved in the study. Male fetal DNA in maternal plasma was detected by using real-time polymerase chain reaction with the SRY gene as a marker. RESULTS: The specificity of the system reached 100% (no Y signal was detected in 17 women pregnant with a female fetus) and the sensitivity reached 100% (SRY amplification in 27 examined samples). CONCLUSIONS: Amplification of free fetal DNA in maternal plasma is a valid and rapid technique for predicting fetal sex in first- and second-trimester pregnancies and could allow the restriction of invasive sampling procedures to male fetuses at risk of X-linked disorders.

Non-invasive fetal sex determination on fetal erythroblasts from the maternal circulation using fluorescence in situ hybridisation.

OBJECTIVE: The main purpose of this study was to evaluate the potential of a non-invasive method for fetal sex determination in clinical practice using dual-colour fluorescence in situ hybridisation (FISH) analysis. We evaluated the differences in nucleated red blood cell (NRBC) recovery from the maternal circulation using various slide preparation procedures following high-gradient magnetic cell separation (double MACS). METHODS: NRBCs were enriched from peripheral blood mononuclear cells of 63 pregnant women between 12 and 37 weeks of gestation by double MACS involving simultaneous CD14+ and CD45+ maternal cell depletion and CD71+ fetal cell enrichment. Isolated cells were analysed by dual-colour FISH with X- and Y-specific probes. Before applying the FISH technique, cells were treated using three different protocols. Cells were either fixed in methanol:acetic acid (3:1) and dropped immediately onto glass slides (protocol 1) or treated with 75 mM KCl before resuspension in fixative (protocol 2). Alternatively, isolated cells were transferred onto glass slides and then treated using a method described in the literature (protocol 3). RESULTS: Using various slide preparation procedures, fixed cell numbers as well as the quality of slides differed significantly. Using protocol 1, fetal sex was well determined in 30 cases, in 15 out of 17 male fetuses (1-13, mean 3 fetal cells were found among 5-164, mean 50 maternal cells) and in 15 female fetuses (7-178, mean 56 fixed cells). On the other hand, interpretation difficulties occurred in 7 out of 8 studied cases using protocol 2 due to a lack of cells or damage to the isolated cells. The highest recovery of fixed cells was achieved using protocol 3 (27-411, mean 186); fetal cells positive for the Y signal (2-12, mean 6) were detectable in all studied cases (n = 16). In 7 of the samples from women carrying female fetuses, we could only detect cells with two X signals (51-182, mean 103). All of the experiments were interpretable due to the presence of compact cells with well-visible red and green signals. CONCLUSION: Our study revealed that using protocol 3 as the post-MACS treatment results in improved NRBC recovery and enables a reliable prospective non-invasive fetal sex determination.

Quantitative analysis of DNA levels in maternal plasma in normal and Down syndrome pregnancies.

BACKGROUND: We investigated fetal and total DNA levels in maternal plasma in patients bearing fetuses affected with Down syndrome in comparison to controls carrying fetuses with normal karyotype. METHODS: DNA levels in maternal plasma were measured using real-time quantitative PCR using SRY and beta-globin genes as markers. Twenty-one pregnant women with a singleton fetus at a gestational age ranging from 15 to 19 weeks recruited before amniocentesis (carried out for reasons including material serum screening and advanced material age), and 16 pregnant women bearing fetuses affected with Down syndrome between 17 to 22 weeks of gestation were involved in the study. RESULTS: The specificity of the system reaches 100% (no Y signal was detected in 14 women pregnant with female fetuses) and the sensitivity 91.7% (SRY amplification in 22 of 24 examined samples). The median fetal DNA levels in women carrying Down syndrome (n=11) and the controls (n=13) were 23.3 (range 0-58.5) genome-equivalents/ml and 24.5 (range 0-47.5) genome-equivalents/ml of maternal plasma, respectively (P = 0.62). The total median DNA levels in pregnancies with Down syndrome and the controls were 10165 (range 615-65000) genome-equivalents/ml and 7330 (range 1300-36750) genome-equivalents/ml, respectively (P = 0.32). The fetal DNA proportion in maternal plasma was 0%-6 % (mean 0.8%) in women carrying Down syndrome and 0%-2.6 % (mean 0.7 %) in the controls, respectively (P=0.86). CONCLUSIONS: Our study revealed no difference in fetal DNA levels and fetal DNA: maternal DNA ratio between the patients carrying Down syndrome fetuses and the controls.

Anti-cyclic citrullinated peptide antibodies in patients with juvenile idiopathic arthritis.

We analysed the presence of anti-cyclic citrullinated peptide (anti-CCP) and anti-keratin (AKA) antibodies of the IgG class in sera of patients with defined juvenile idiopathic arthritis (JIA) of various subgroups with more than one year duration of the disease. Enzyme-linked immunosorbent assay (Immunoscan RA, Eurodiagnostica, The Netherlands) and an indirect immunofluorescence (IIF) test on rat oesophagus substrate (ImmuGloTM, Immco Diagnostics, Buffalo, USA) were used for the detection and quantification of anti-CCP and AKA antibodies in 140 patients with JIA (64 male and 76 female) aged 2-47 years (median 16.5 years). Overall, anti-CCP were found in 7/140 (5.0%) patients including 3/52 RF negative polyarthritis, 2/18 RF positive polyarthritis, 1/15 enthesitis related arthritis and 1/5 unclassifiable arthritis. AKA were detected in 40/140 patients (28.6%, p = 0.04) including 2/11 systemic arthritis, 2/32 oligoarthritis, 18/52 patients with RF negative polyarthritis (34.6%, p = 0.01), 14/18 RF positive polyarthritis (77.8%, p = 0.000002), 2/15 enthesitis related arthritis and 2/3 psoriatic arthritis. While simultaneous negativity for AKA and anti-CCP occurred in most (97/140; 69.3%) studied cases, simultaneous antibody positivity was found only in few (4/140; 2.9%) studied samples. We conclude that while AKA measured using IIF on rat esophagus can be detected approximately in one third of patients with definite JIA with more than 1 year duration of the disease, only rare occurrence of anti-CCP was observed. We conclude that AKA seem to be partly useful to confirm JIA diagnosis, however, useless to follow-up severity or activity in JIA patients. Anti-CCP do not have any additional value in MA cohort in comparison to RA where their diagnostic and prognostic importance was reported.