Optimization of the fluorescence in situ hybridization (FISH) technique for high detection efficiency of very small proportions of target interphase nuclei.

Using commercially available fluorochrome-labeled probes specific for chromosomes X, Y, 13, 18, and 21, we optimized the technical protocols for fluorescence in situ hybridization (FISH) so that the highest sensitivity and specificity were achieved. Also, we compared the optical properties of different types of fluorescent labels in an effort to develop the most efficient FISH protocol, including the determination of which types of labels are the easiest to count accurately. The lymphocytes were purified from blood of normal male and female newborns, normal male and female adults, and a trisomy 21 male adult. Male and female lymphocytes were mixed in five different combinations. For each combination, the male lymphocytes either from newborns or from adults were diluted with female lymphocytes in seven different proportions. For each of these 35 different cell mixtures, 100,000 nuclei were analyzed and scored in a blind fashion. Among the different fluorochrome-labeled probes, the highest sensitivity and specificity were achieved when SpectrumAqua CEP-Y/SpectrumOrange CEP X probe mixture, SpectrumAqua CEP-18, SpectrumOrange LSI-13, and SpectrumOrange LSI-21 were hybridized. The hybridization sensitivity and specificity were higher than 99% for the identification of chromosomes X, Y, 13, and 18, and higher than 98% for the detection of trisomy 21. The proportion of false-positive signals was under 0.005% for XY detection and lower than 0.14% for autosome detection. With these high hybridization sensitivities and specificities, the optimized FISH protocol developed in our laboratory has the potential to detect very rare events, e.g., when the proportion of cells being sought is lower than 0.01%. In other words, our protocol allows the specific detection of one male cell sunken among 10,000 female cells.

A prospective longitudinal study of platelet angiotensin II receptors for the prediction of preeclampsia.

OBJECTIVES: We developed a simplified method using a relatively small volume of blood for the determination of platelet angiotensin II receptors by saturation analysis and we evaluated its performance for the prediction of preeclampsia. DESIGN AND METHODS: A platelet suspension with minimal contamination by leukocytes and erythrocytes is obtained by centrifugation and washing. The platelet concentrate is incubated in a multi-well plate with increasing concentration of radiolabelled angiotensin II in the presence or absence of an excess of unlabelled angiotensin II. Bound and free fractions are separated using an oil mixture. Maximum binding is determined by Scatchard plot. This method was compared with a previously reported method. Our method was prospectively evaluated in 801 women attending our institution for routine prenatal care. A specimen was obtained at each trimester of pregnancy whenever possible. Diagnosis of preeclampsia was done postnatally by an experienced obstetrician. RESULTS: The method showed acceptable correlation with a previously published method although a proportional bias of 2.1 was observed between the two methods. No differences in mean maximum binding were observed between normal and affected pregnancies at either trimester. Even when the results were analyzed longitudinally, using the change in maximum binding between two trimesters for each patient, no significant increase could be documented in preeclamptic pregnancies. CONCLUSIONS: Platelet angiotensin II receptor measurement is not a clinically useful marker for the prediction of preeclampsia.

Effect of repeated freeze-thaw cycles on maternal serum biological markers for the detection of fetal trisomies.

OBJECTIVES: We studied the stability of maternal blood markers for screening for Down syndrome (alpha-fetoprotein, unconjugated estriol, intact human chorionic gonadotropin (hCG) and free beta-human chorionic gonadotropin) upon repeated freeze-thaw cycles. DESIGN AND METHODS: Forty-three samples collected from second trimester normal pregnancies were submitted to five freeze-thaw cycles. After each cycle, the markers were measured using kits and instruments from Wallac Canada (AutoDelfia). Results were compared by repeated measures analysis of variance and by analysis of linear trend (after mathematical transformation of the results in order to decrease between-sample variation) as a function of the number of freeze-thaw cycles. RESULTS: No significant differences were observed by ANOVA (p > 0.1) for any marker. Intact hCG showed a statistically significant linear downward trend (slope = -0.0063, p = 0.009) while free beta-hCG increased (slope = 0.011, p = 0.004). After five freeze-thaw cycles, a mean decrease of 3.2% is predicted for intact hCG while free beta-hCG would increase by 5.5% on average. CONCLUSION: We conclude that the studied markers do not show clinically significant changes under the evaluated conditions. The observed changes of intact hCG and free beta-hCG would have a limited impact on the screening performance.