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

Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry

Various fixation and permeabilization techniques have been developed for detection of intracellular antigens by flow cytometry; however, there are few studies using flow cytometry to detect the frequency of intracellular nucleic acids, particularly RNA. We tested six different permeabilization methods in order to gain access to a high quality method with minimal damage to intracellular components focusing on 18S rRNA in HeLa cells. HeLa cells were fixed in 2% paraformaldehyde. A variety of detergents and enzymes including saponin, TritonX-100, Tween-20, NP40, Proteinase K, and streptolysin O were used to optimize a protocol of permeabilization for the flow cytometric enumeration of intracellular 18S rRNA. Treated cells were subjected to standard protocol of flow cytometric in situ hybridization in the presence of FITC-labeled sense and antisense probes to detect 18S ribosomal RNAs. Samples were then analyzed on a FACSCalibur flow cytometer. To evaluate cell morphology, following hybridization the cells were fixed on glass slide, covered with DAPI, and evaluated on a fluorescent microscope with appropriate filter sets. In comparison with other methods, maximum cell frequency in percentage and fluorescent intensity [M1=2.1%, M2=97.9%] were obtained when the cells were treated with 0.2% Tween-20 and incubated for 30 min [p=0.001]. Our study indicated that the highest levels of mean fluorescence could be obtained when the cells were treated with Tween-20. However, it should be taken into consideration that for a successful flow cytometric result, other interfering factors such as hybridization conditions should also be optimized

Gibbon ape leukemia virus transduction of peripheral blood CD34+-derived dendritic cells

Dendritic cells [DCs] play a critical role in the immune response and are a candidate for immunotherapy in cancer. Since gibbon ape leukemia virus [GALV] transduction of CD34+ cells is reasonably efficacious, we assessed the efficacy of GALV transduction of CD34+ derived DCs as a possible approach to creating genetically modified DCs for immunotherapy. Peripheral blood CD34+ cells were transduced with retroviruses obtained from the PG13/LN C8 cell line, with the neomycin gene as a marker gene. After prestimulation of hematopoietic cells for 24 hours with 10 ng/mL interleukin [IL]-3, 10 ng/mL IL-6, 100 ng/mL stem cell factor, 100 ng/mL granulocyte-macrophage colony stimulating factor and 8 micro g/mL protamine sulfate, the cells were cultured in a transforming media prior to differentiating into DCs by GM-CSF, TNF-alpha and IL-4. Immunophenotyping analyses for confirmation of the generated DCs, colony formation assay and PCR were done for the expression of neomycin gene in the transduced cells. Titration of viral vectors indicated a transduction efficiency of 1 x10[5] CFU/mL Transduction efficiency for the CD34+ cells transformed to DCs was 45% and 38% before and after DC differentiation, respectively. Additionally, a mean [SEM] of 26.9% [11.4%] and 41.4% [11.8%] of the genetically modified DCs were positive forCD86+ HLA-DRand CD1alpha+CD14, respectively. This study showed that the majority of transduced CD34+ cells were successfully differentiated into cells identical to DCs according to morphology and immunophenotyping features, which could be a potential application in immunotherapy