Development of flow cytometry-fluorescent in situ hybridization [Flow-FISH] method for detection of PML/RARa chromosomal translocation in acute promyelocytic leukemia cell line

Background: acute Promyelocytic Leukemia [APL] is a subclass of acute myeloid leukemia. The chromosomal aberration in 95% of APL cases is t[15; 17] [q22; q21], which prevents cell differentiation. Characterization of the underlying molecular lesion is valuable in determining optimal treatment strategy. The goal of this study was to develop a new and powerful Flow- FISH technique to detect the long isoform [L] of PML-RARa fusion transcript in NB4 cell line

Methods: to achieve the best condition for fixation, two different fixatives including 2% paraformaldehyde and 75% ethanol were used. 0.2% Triton X-100 and 0.2% saponin were used for the permeabilization step .In hybridization, a wide range of times and temperatures were used and probe was designed in FRET system. Results were confirmed by fluorescent microscope assay and reverse transcription PCR

Results: in the present study, a novel technique was successfully optimized that combines in situ hybridization with flow cytometry to detect the presence of PML-RARa transcript. Using standard fixation and permeabilization protocol of 2% PFA and 0.2% saponin gave the best fluorescent results in flow cytometry. Also, results indicated that the optimum time and temperature for hybridization was 2 hr at 42degreeC. The results of reverse transcription PCR and fluorescent microscopy confirmed the presence of PML-RARa transcript

Conclusion: the concordance between the results of Flow-FISH and those of two other techniques including reverse transcription PCR and FISH indicated that this method would be applicable as a diagnostic test for APL in clinical samples and MRD monitoring

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

Chimeric external control to quantify cell free DNA in plasma samples by real time PCR

Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA [cfDNA] is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control [EDC] is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for monitoring stability of cfDNA concentration in blood samples over time

Methods: A DNA fragment of 167 bp chimeric sequence of parvovirus B19 and pBHA designated as EDC fragment was designed. To determine the impact of different factors during DNA extraction processing on quantification of cfDNA, blood samples were collected from normal subjects and divided into aliquots with and without specific treatment. In time intervals, the plasma samples were isolated. The amplicon of 167 bp EDC fragment in final concentration of 1.1 pg/ 500 microl was added to each plasma sample and total DNA was extracted by an in house method. Relative and absolute quantification real time PCR was performed to quantify both EDC fragment and cfDNA in extracted samples

Results: Comparison of real time PCR threshold cycle [Ct] for cfDNA fragment in tubes with and without specific treatment indicated a decrease in untreated tubes. In contrast, the threshold cycle was constant for EDC fragment in treated and untreated tubes, indicating the difference in Ct values of the cfDNA is because of specific treatments that were made on them

Conclusions: Spiking of DNA fragment size relevant to cfDNA into the plasma sample can be useful to minimize the bias due to sample preparation and extraction processing. Therefore, it is highly recommended that standard external DNA control be employed for the extraction and quantification of cfDNA for accurate data analysis

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 foundusing 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

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