Expression of Basophil Activation Markers in Pediatric Asthma.

BACKGROUND: Asthma is very common in children and its diagnosis is based on clinical manifestations, which can be misdiagnosed as other respiratory diseases with similar signs and symptoms. OBJECTIVE: To analyze the expression of ST2L and CD203c in the diagnosis of pediatric asthma. METHODS: Basophils were purified from whole blood samples of patients and healthy controls using Ficol-Paque gradient and Basophil Isolation Kit. RNA extraction was done by RNX-Plus solution and after synthesis of cDNA, the gene expression was analyzed by means of real time PCR. RESULTS: Patients expressed significantly higher levels of CD203c than healthy controls (p=0.01). Although there was an increase in the transcription level of ST2L gene in patients, the results were not statistically significant compared to those obtained from the healthy controls (p>0.05). A Specificity of 60% and a sensitivity of 73% were found using ROC curve for CD203c expression. Patients with positive family history of asthma exhibited more CD203c and ST2L expression (p<0.05). CONCLUSION: It is proposed that determining CD203c expression by real time PCR may be an effective technique for diagnosis of pediatric asthma.

Fetal RHD Genotyping Using Real-Time Polymerase Chain Reaction Analysis of Cell-Free Fetal DNA in Pregnancy of RhD Negative Women in South of Iran.

BACKGROUND: Maternal-fetal RhD antigen incompatibility causes approximately 50% of clinically significant alloimmunization cases. The routine use of prophylactic anti-D immunoglobulin has dramatically reduced hemolytic disease of the fetus and newborn. Recently, fetal RHD genotyping in RhD negative pregnant women has been suggested for appropriate use of anti-D immunoglobulin antenatal prophylaxis and decrease unnecessary prenatal interventions. MATERIALS AND METHODS: In this prospective cohort study, in order to develop a reliable and non-invasive method for fetal RHD genotyping, cell free fetal DNA (cffD- NA) was extracted from maternal plasma. Real-time quantitative polymerase chain reaction (qPCR) for detection of RHD exons 7, 5, 10 and intron 4 was performed and the results were compared to the serological results of cord blood cells as the gold standard method. SRY gene and hypermethylated Ras-association domain family member 1 (RASSF1A) gene were used to confirm the presence of fetal DNA in male and female fetuses, respectively. RESULTS: Out of 48 fetuses between 8 and 32 weeks (wks) of gestational age (GA), we correctly diagnosed 45 cases (93.75%) of RHD positive fetuses and 2 cases (4.16%) of the RHD negative one. Exon 7 was amplified in one sample, while three other RHD gene sequences were not detected; the sample was classified as inconclusive, and the RhD serology result after birth showed that the fetus was RhD-negative. CONCLUSION: Our results showed high accuracy of the qPCR method using cffDNA for fetal RHD genotyping and implicate on the efficiency of this technique to predict the competence of anti-D immunoglobulin administration.

Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum.

BACKGROUND: Discovery of short cell free fetal DNA (cffDNA) fragments in maternal plasma has created major changes in the field of prenatal diagnosis. The use of cffDNA to set up noninvasive prenatal test is limited due to the low concentration of fetal DNA in maternal plasma therefore, employing a high efficiency extraction method leads to more accurate results. The aim of this study was to evaluate the efficiency of Triton/Heat/Phenol (THP) protocol in comparison with the QIAamp DNA Blood mini Kit for cffDNA purification. METHODS: In order to evaluate the efficiency of THP protocol, DNA of Rhesus D (RhD) negative pregnant women's plasma was collected, then real-time PCR for RHD exon 7 was performed. The Ct value data of real time PCR obtained by two different methods were compared and after delivery serology test on cord blood was done to validate the real time PCR results. RESULTS: The results indicated significant differences between two extraction methods (p=0.001). The mean±SD of Ct-value using THP protocol was 33.8±1.6 and 36.1±2.47 using QIAamp DNA Blood mini Kit. CONCLUSION: Our finding demonstrated that THP protocol was more effective than the QIAamp DNA Blood mini Kits for cffDNA extraction and lead to decrease the false negative results.