Quantitative Fluorescent Research Articles

Rapid Detection of Sex Chromosomal Aneuploidies by QF-PCR: Application in 200 Men with Severe Oligozoospermia or Azoospermia

Klinefelter syndrome is the most common genetic cause of severe male factor infertility. Cytogenetic evaluation of metaphase chromosomes generally has a long turnaround time. We describe a reliable molecular genetic method that can be completed in 2 working days to identify the presence of any extra X chromosomes. The quantitative fluorescent (QF) 5-plex PCR includes the amplification of amelogenin, which is present on both sex chromosomes in a biallelic form, a polymorphic short tandem repeat (STR) on the pseudoautosomal region of X and Y (X22), two polymorphic X-specific STRs (DXS6803, DXS6809), and a Y-specific marker (SY134), in a single tube. The presence of an extra X chromosome is recognized either by a supernumerary peak or an increased peak area based on criteria we have developed. The application of the method on 200 patients resulted in the identification of 14 patients (7%) with Klinefelter syndrome or a variant form (2 SRY-positive 46,XX men), as well as an additional patient with 47,XYY karyotype. The QF-PCR method, along with Y chromosome microdeletion testing, can be used as a first-step genetic analysis in azoospermic or severely oligozoospermic patients for the rapid identification of sex chromosome aneuploidies.

Prenatal Diagnosis of Common Aneuploidies in Transcervical Samples Using Quantitative Fluorescent-PCR Analysis

The aim of this study was to test the feasibility of diagnosing common fetal chromosomal aneuploidies using quantitative fluorescent (QF)-PCR on transcervical cell (TCC) samples collected in the first trimester of pregnancy by means of intrauterine lavage (IUL). A total of 181 TCC samples were retrieved from pregnant women between 5 and 12 weeks of gestation, immediately before elective termination of pregnancy, at which time corresponding placental tissue and maternal blood specimens were also obtained. Isolation of trophoblastic cells by micromanipulation was attempted in all TCC samples. Micromanipulated specimens were analyzed by multiplex QF-PCR, including short tandem repeats for the chromosomes X, Y, 21, 18, and 13. The micromanipulation was successful in 152 of 181 cases (84.8%) where chorionic villous filaments and/or cell clumps of seeming trophoblastic origin could be isolated. All 152 samples were tested by QF-PCR analysis and peaks of paternal origin could be documented in all cases. Two cases of trisomy 21 and two cases of monosomy X0 were detected by means of QF-PCR assay, in accordance with the results obtained in corresponding placental samples. This study provides evidence that the use of multiplex QF-PCR amplification of selected microsatellites could be applied to micromanipulated TCC samples and in particular to IUL samples, which often contain trophoblastic cells, for the detection of chromosomal aneuploidies. The approach described in this study appears, therefore, a very promising tool toward non-invasive prenatal genetic diagnosis in the early stage of gestation.

The Dynamics of the Rapsyn Scaffolding Protein at Individual Acetylcholine Receptor Clusters

Rapsyn, a cytoplasmic receptor-associated protein, is required for the clustering of acetylcholine receptors (AChRs). Although AChR dynamics have been extensively studied, little is known about the dynamics of rapsyn. Here, we used a rapsyn-green fluorescent protein (GFP) fusion protein and quantitative fluorescent imaging to study the dynamics of rapsyn in transfected C2C12 myotubes. First, we found that rapsyn-GFP expression at clusters did not alter AChR aggregation, function, or turnover. Quantification of rapsyn immunofluorescence indicated that the expression of rapsyn-GFP proteins at clusters does not increase the overall rapsyn density compared with untransfected myotube clusters. Using time lapse imaging and fluorescence recovery after photobleaching, we demonstrated that the recovery of rapsyn-GFP fluorescence at clusters was very fast, with a halftime of about approximately 1.5 h (approximately 3 times faster than AChRs). Inhibition of protein kinase C significantly altered receptor insertion, but it had no effect on rapsyn insertion. When cells were treated with the broad spectrum kinase inhibitor staurosporine, receptor insertion was decreased even further. However, inhibition of protein kinase A had no effect on insertion of either rapsyn or receptors. Finally, when cells were treated with neural agrin, rapsyn and AChRs were both directed away from preexisting clusters and accumulated together in new small clusters. These results demonstrate the remarkable dynamism of rapsyn, which may underlie the stability and maintenance of the postsynaptic scaffold and suggest that the insertion of different postsynaptic proteins may be operating independently.

Use of the Quantitative Fluorescent-PCR Assay in the Study of Fetal DNA from Micromanipulated Transcervical Samples

The purpose of this study was to evaluate the validity of the combined use of micromanipulation and quantitative fluorescent (QF)-PCR assay for the identification of fetal elements in transcervical cell (TCC) samples collected in early pregnancy. TCC samples were obtained by intrauterine lavage (IUL) in 113 pregnant women who were between 7 and 12 weeks pregnant before termination of pregnancy. All IUL samples were screened under an inverted microscope, at which time the isolation of fetal cells by micromanipulation was attempted. QF-PCR assay, using 9 small tandem repeat (STR) markers for chromosomes 13, 18, 21, X, and Y, was performed in all specimens to identify fetal cells in TCC samples and the corresponding placental tissue and blood specimens. TCC samples from male fetuses in which either the micromanipulation or QF-PCR analysis were unsuccessful, were studied with fluorescent in situ hybridization (FISH), using probes for X and Y chromosomes. Isolation of supposed fetal material from IUL samples was carried out by means of micromanipulation in 93 cases (82.3%), where discernible chorionic villous filaments or cell clumps of probable trophoblastic origin were present. The QF-PCR analysis was performed in all 93 IUL samples and paternal peaks could be documented in 88 cases (94.6%) thus confirming the presence of fetal cells. Thirteen cases negative to micromanipulation and derived from male fetuses and four male cases not informative with QF-PCR analysis, after micromanipulation, were then tested with FISH assay using probes for sexual chromosomes. In six samples, rare (2-3%) male fetal cells were detected. Considering the combined results obtained from QF-PCR and FISH assays, the overall fetal sexing was correct in 83.2% of cases (94 of 113). This study provides evidence that fetal cells are present in a high proportion of IUL samples. Micromanipulation appears to be an extremely efficient method for the isolation of trophoblastic elements. This study also confirms the potential of IUL as a possible alternative to the traditional prenatal diagnostic procedures for the recovery of fetal cells in precocious stage of gestation, and validates the combination of the isolation of such fetal elements by means of micromanipulation and analysis with the QF-PCR assay for the identification of the most frequent prenatal chromosomal aneuploidies.

Real-Time Quantitative Fluorescent Reverse Transcriptase-PCR for Detection of Severe Acute Respiratory Syndrome-Associated Coronavirus RNA

Aim: SARS-associated coronavirus (SARS-CoV) has been confirmed as the pathogen for severe acute respiratory syndrome (SARS). The aim of our study was to construct a sensitive and specific real-time quantitative fluorescent (QF) reverse transcriptase (RT)-PCR method for the detection of SARS-CoV RNA. Methods: Stored blood specimens from 44 patients with confirmed SARS were used along with blood samples from two sets of controls, 30 healthy volunteers who had no contact with SARS patients, and 30 healthy doctors and nurses who had contact with SARS patients but were without symptoms of SARS. Two pairs of primers were synthesized by the Shanghai Sangon Company according to SARS-CoV BJ01 strain sequence (AY278488), and then a pair of primers were designed and compared with a pair of primers published by WHO. Results: Using serial dilutions of SARS-CoV, the 44 blood samples from SARS patients specimens were tested. Using a 0.01% dilution of SARS-CoV, all 44 clinical samples tested positive in our assay. In comparison, using a 0.1% dilution of SARS-CoV, 26 of the 44 samples tested positive using the WHO primers. In the QF-RT-PCR assay, there was a linear amplification from 100 copies to 108 copies of the control RNA per RT-PCR and at least 10 copies, and sometimes even 1 copy, of target RNA tested positive in our assay. Conclusion: The primer we developed is sufficiently sensitive and specific to diagnose symptomatic SARS-CoV infections and for monitoring virus load.

Strategies for the isolation and detection of fetal cells in transcervical samples.

The aim of the study was to evaluate the detection of fetal cells from transcervical samples, collected in early pregnancy, by means of different molecular techniques. The value of the isolation of trophoblasts using an inverted microscope, also referred to as micromanipulation, is discussed. All the 89 specimens were obtained by intrauterine lavages before termination of pregnancy (TOP), between 7 and 12 weeks of gestation. Micromanipulation was carried out in a subgroup of 57 for the isolation of fetal material. Fetal sexing was achieved by FISH (fluorescent in situ hybridisation) using fluorescently labelled probes for X and Y chromosomes and by polymerase chain reaction (PCR). Male samples were also investigated for aneuploidy of the chromosome 21. Quantitative fluorescent (QF)-PCR using two short tandem repeat (STR) markers for chromosome 21 was carried out in 26 micromanipulated samples. FISH analysis revealed that 45/89 placental samples derived from pregnancies with male fetuses. Correct sexing of the lavage samples from male pregnancies was achieved in 41/45 (91%) using dual-FISH technique, and in 43/45 (95.5%) with PCR. All the samples derived from male pregnancies tested for chromosome 21 were normal. From 57 samples subjected to micromanipulation, 51 (89.5%) showed discernible chorionic villous filaments or cell clumps of possible trophoblastic origin. One case of tetraploidy and two cases of monosomy were recorded. The rate of fetal cells, in the non-micromanipulated samples, was between 4% and 97% (mean 54.3%). In micromanipulated specimens, maternal contaminant cells were absent or extremely rare (1-2%). The efficiency of the QF-PCR analysis in detecting paternal peaks in all lavage samples was only 61.5%. The present study confirms the presence of fetal cells in a very high proportion of both whole and micromanipulated intrauterine lavage samples. The isolation of trophoblastic elements can be achieved in most cases by micromanipulation. FISH and PCR techniques allowed the analysis of the most common fetal aneuploidies, confirming the power of this minimally invasive method.

Detection of fetal cells in intrauterine lavage samples collected in the first trimester of pregnancy.

The aim of the present study was first to evaluate the presence of fetal cells in transcervical cell (TCC) samples collected by intrauterine lavage in the first trimester of pregnancy, and then to compare different methods for the detection of these cells. TCC samples were collected by intrauterine lavage before termination of pregnancy (TOP) from 81 pregnant women between 7 and 12 weeks of gestation. Samples of placental tissue were collected from each patient at TOP, whereas maternal peripheral blood samples were obtained in 57 cases. DNA extracted from 81 lavage and the corresponding placental samples was amplified by a polymerase chain reaction (PCR) assay using primers for SRY and HUMARA genes. All 81 lavage samples were also analysed by fluorescent in situ hybridisation (FISH) using direct-labelled probes for X chromosome alpha-satellite (DXZ1, Xp11.1-q11.1) and Y chromosome alpha-satellite (DYZ3, Yp11.1-q11.1) regions. In 57 cases, a quantitative fluorescent (QF) PCR assay, involving the use of two small tandem repeat (STR) markers (D21S11, D21S14.11) specific to chromosome 21 was employed to analyse DNA extracted from placental tissue, lavage and maternal blood samples. PCR analysis revealed that 40/81 placental samples were from male pregnancies. Correct sexing was achieved with the PCR technique in 30/40 (75%) lavage samples retrieved from pregnant women with male conceptuses and in all 41 (100%) samples collected from pregnancies with female fetuses. With the FISH analysis, nuclei bearing X and Y signals were observed in 32/40 cases (80%) from known male pregnancies, the rate of fetal cells ranging between 2% and 95%, whereas nuclei showing X and Y signals were not detected in any of the 41 lavage samples from known female pregnancies. Paternal peaks were present in 30/57 (52.6%) lavage samples tested by QF-PCR. The results suggest that fetal cells can be found, at a significant rate, in a very high proportion of intrauterine lavage samples. Therefore, this sampling technique can be regarded as a promising tool towards minimally invasive prenatal diagnosis. The FISH and PCR methods showed a similar efficiency in detecting fetal cells.

Impact of unified procedures as implemented in the Canadian Quality Assurance Program for T lymphocyte subset enumeration

The objective of the Canadian Quality Assurance Program (CQAP) is to provide the most reproducible and accurate T-cell subset enumeration for individuals living with HIV who are enrolled in the Canadian Clinical Trial Network for Human Immunodeficiency Virus (HIV) and Acquired Immune Deficiency Syndrome (AIDS) Therapies (abbreviated as CTN). The Canadian National Laboratory for Analytical Cytology, within the Laboratory Centre for Disease Control, is part of the Health Protection Branch of Health Canada. For the past eight years, the Laboratory for Analytical Cytology has been responsible for delivering a bilingual quality assurance program for CD4 T-cell enumeration. This federal program, which integrates biotechnology transfer with quality assessment, was achieved through the organization of workshops focused on technology transfer and essential skill-building techniques. Two training sessions were conducted for the CTN flow cytometer operators. The first introduced the concept of window of analysis, to demonstrate the practical benefits of unified quantitative fluorescent measurement. As a follow-up to the first workshop, participants performed a series of quantitative assays that monitored the expression of CD69, an early activation marker. This quantitative fluorescence protocol was performed with acceptable inter-laboratory variation using modified commercial kits. The second workshop focused on a absolute count method based on a single platform. Four preserved whole-blood preparations were tested with this approach. The combined effort reduced inter-laboratory variation. The direct impact was monitored as related to the frequency of participation. Over the years, the standard deviation of average accumulated variation decreased dramatically with increased frequency of participation, from 10% to <4%. Cytometry 33:146–155, 1998. © 1998 Wiley-Liss, Inc.

Impact of unified procedures as implemented in the Canadian Quality Assurance Program for T lymphocyte subset enumeration

The objective of the Canadian Quality Assurance Program (CQAP) is to provide the most reproducible and accurate T-cell subset enumeration for individuals living with HIV who are enrolled in the Canadian Clinical Trial Network for Human Immunodeficiency Virus (HIV) and Acquired Immune Deficiency Syndrome (AIDS) Therapies (abbreviated as CTN). The Canadian National Laboratory for Analytical Cytology, within the Laboratory Centre for Disease Control, is part of the Health Protection Branch of Health Canada. For the past eight years, the Laboratory for Analytical Cytology has been responsible for delivering a bilingual quality assurance program for CD4 T-cell enumeration. This federal program, which integrates biotechnology transfer with quality assessment, was achieved through the organization of workshops focused on technology transfer and essential skill-building techniques. Two training sessions were conducted for the CTN flow cytometer operators. The first introduced the concept of window of analysis, to demonstrate the practical benefits of unified quantitative fluorescent measurement. As a follow-up to the first workshop, participants performed a series of quantitative assays that monitored the expression of CD69, an early activation marker. This quantitative fluorescence protocol was performed with acceptable inter-laboratory variation using modified commercial kits. The second workshop focused on a absolute count method based on a single platform. Four preserved whole-blood preparations were tested with this approach. The combined effort reduced inter-laboratory variation. The direct impact was monitored as related to the frequency of participation. Over the years, the standard deviation of average accumulated variation decreased dramatically with increased frequency of participation, from 10% to <4%.

Automated quantitative fluorescent image analysis of cervical cytology

Cervical cytology from 122 patients with histories of one or more abnormal Pap smears were studied using slide-based automated quantitative fluorescence image analysis (QFIA) in order to determine the usefulness of the QFIA technique in detecting neoplasias. The purpose of the study was to evaluate the accuracy and validity of QFIA as a cytological method for the early detection of cervical cancer, based on comparisons with the conventional Pap smear and histopathology. The results of the analysis demonstrated that the sensitivity of the automated QFIA cytology was 100% for high-grade neoplasms (CIN III and invasive carcinoma), compared with 82% for the Pap smear. Evaluation of lower grade neoplasms was also performed. Initial analyses of the data indicate that QFIA, which uses objective biochemical criteria to identify cells with increased nuclear nucleic acid content, is a more sensitive detector of cervical neoplasia of all grades than is conventional Pap cytology.