PCR Signal Research Articles

Potential wildlife sentinels for monitoring the endemic spread of human buruli ulcer in South-East australia.

The last 20 years has seen a significant series of outbreaks of Buruli/Bairnsdale Ulcer (BU), caused by Mycobacterium ulcerans, in temperate south-eastern Australia (state of Victoria). Here, the prevailing view of M. ulcerans as an aquatic pathogen has been questioned by recent research identifying native wildlife as potential terrestrial reservoirs of infection; specifically, tree-dwelling common ringtail and brushtail possums. In that previous work, sampling of environmental possum faeces detected a high prevalence of M. ulcerans DNA in established endemic areas for human BU on the Bellarine Peninsula, compared with non-endemic areas. Here, we report research from an emergent BU focus recently identified on the Mornington Peninsula, confirming associations between human BU and the presence of the aetiological agent in possum faeces, detected by real-time PCR targeting M. ulcerans IS2404, IS2606 and KR. Mycobacterium ulcerans DNA was detected in 20/216 (9.3%) ground collected ringtail possum faecal samples and 4/6 (66.6%) brushtail possum faecal samples. The distribution of the PCR positive possum faecal samples and human BU cases was highly focal: there was a significant non-random cluster of 16 M. ulcerans positive possum faecal sample points detected by spatial scan statistics (P<0.0001) within a circle of radius 0.42 km, within which were located the addresses of 6/12 human cases reported from the area to date; moreover, the highest sample PCR signal strength (equivalent to ≥106 organisms per gram of faeces) was found in a sample point located within this cluster radius. Corresponding faecal samples collected from closely adjacent BU-free areas were predominantly negative. Possums may be useful sentinels to predict endemic spread of human BU in Victoria, for public health planning. Further research is needed to establish whether spatial associations represent evidence of direct or indirect transmission between possums and humans, and the mechanism by which this may occur.

Evaluation Of IKZF1 Δ4-7 Deletion As a Suitable Marker For Minimal Residual Disease Monitoring; A Study Of 161 Consecutive Acute Lymphoblastic Leukaemia (ALL) Patients On The On-Going UKALL14 Trial

Minimal residual disease (MRD) testing is vital to risk assignment in acute lymphoblastic leukaemia (ALL). Quantification of patient-specific rearrangements of immunoglobulin and T-cell receptor (Ig/TCR) genes is the most standardised method in current use. Recent studies demonstrated that deletion in the IKZF1 gene is common and is prognostic of poor outcome. IKZF1 targets could offer potential for MRD monitoring in BCR-ABLnegative (neg) ALL. The 5' and 3' break points of this deletion are highly conserved, making it possible to design a universal PCR assay to amplify the fusion genomic sequence created by the deletion.With the initial aim of developing an MRD assay we studied the incidence of IKZF1 Δ 4-7 in 161 consecutive patients enrolled on the on-going adult ALL trial UKALL14, aged 25-59 years. We carried out PCR using breakpoint-specific primers designed as close as possible to the breakpoint. In order to ensure that the screening PCR enabled sensitive detection of the deletions, serial dilution of a positive control SUP-B15 cell line diluted into non-tumoral DNA was analysed. IKZF1 Δ 4-7 could be detected with a sensitivity of at least 10-4 (0.01%). PCR bands could be readily allocated to “strong” or “weak” on visual inspection; all PCR positive signals were confirmed as IKZF1 Δ 4-7 by Sanger sequencing of the PCR products and the breakpoints mapped. The frequency of the deletion in the total population studied was 80/161 (50%); 23/35 (66%) in the BCR-ABL positive (pos) subgroup and strikingly 57/126 (45%) in the BCR-ABLneg subgroup. The high rate of IKZF1 Δ 4-7 in the BCR-ABLneg patients was unexpected, being approximately double the reported incidence BCR-ABLneg adults.Real time quantitative (RQ)-PCR analyses to investigate the suitability of IKZF1 quantitation as the basis for an MRD assay was performed on 26 BCR-ABLneg cases, selected only on the basis of availability of follow-up material. Assays were assessed using the same criteria applied by EuroMRD for Ig/TCR quantification. On that basis, “limited testing” of PCR amplifications at the 10-2 (1%) and 10-4(0.01%) dilution points was performed on all 26 samples. To our surprise, only 5 samples gave acceptable data to qualify for a quantitative MRD assay. Notably, all the 21 cases that failed “limited testing” had yielded a weak PCR signal on initial screening, suggesting the PCR assay have detected intragenic deletions restricted to minor subclones.Although subclonal IKZF1 gene alterations are well described, the apparent high frequency of these events in our cohort (21/26 tested for RQ-PCR assay) was surprising. To further analyse this, we performed MRD-based genomic quantification of all 26 diagnostic specimens using SUP-B15 dilution series. The percentage of ALL cells bearing IKZF1 Δ 4-7 in the putative 21 subclonal cases was <0.01% compared to >90% in the putative 5 major clonal cases. In total, 82% of BCR-ABLneg cases gave a weak PCR band at initial screening, suggesting that most of the Δ 4-7 in BCR-ABLneg adult ALL are subclonal.In the 5 cases where a quantifiable IKZF1 Δ 4-7-based MRD assay could be developed, MRD analysis was performed on 12 follow-up samples and sensitivity and quantitative range of at least 10-4 (0.01%) and 5×10-4 (0.05%) were obtained, respectively. These parameters compare favourably with the performance of standard Ig/TCR assays. Notably in one sample MRD could only be quantified by the IKZF1 deletion approach (level= 7.79E-05, ∼ 0.008%).IKZF1 lesions are not leukaemia-initiating events, hence the stability of this alteration during the history of ALL is not clear. To address this, 14 diagnosis and relapse paired samples were analysed. Three different patterns emerged: in 5 cases the IKZF1 Δ 4-7 at diagnosis was preserved at relapse; in 3 cases the lesion was newly acquired at relapse; and in 3 cases the deletion was lost at relapse, notably all these 3 cases had showed a weak PCR signal, suggesting the deletion which was lost at relapse was subclonal at diagnosis. Theoretically this observation suggests that IKZF1deletion-based MRD monitoring carries a risk of missing a resurgent clone.In conclusion, IKZF1 Δ 4-7 can provide highly sensitive MRD assays and could be a potential candidate to add to the repertoire of currently available MRD markers. However, we have shown limited applicability due to many IKZF1 deletions occurring in putative subclones. The stability of these deletions in major clones remains to be determined. Disclosures:No relevant conflicts of interest to declare.

Comparison of Human Papillomavirus Detections in Urine, Vulvar, and Cervical Samples from Women Attending a Colposcopy Clinic

While urine-based sampling for human papillomavirus (HPV) is being explored as a simple and noninvasive approach for cervical cancer screening, data comparing HPV genotyping in urine and those in cellular sampling of the cervix and vulva, and their correlation with rigorously confirmed cervical disease status, are sparse. We performed HPV genotyping on voided-urine and clinician-collected vulvar and cervical samples from 72 women undergoing colposcopy. Although urine-based HPV carcinogenic HPV detection was lower (58.3%) than cervical (73.6%) and vulvar (72.1%) detection (P = 0.05 and 0.07, respectively), the agreement of urine HPV with cervical and vulvar HPV was moderate (kappa = 0.55) and substantial (kappa = 0.62), respectively. Urine-based carcinogenic HPV detection had a clinical sensitivity of 80.8% (95% confidence interval [CI] = 60.7 to 93.5) and a specificity of 53.3% (95% CI = 37.9 to 68.3) for diagnosing cervical intraepithelial neoplasia grades 2/3 (CIN2/3) on histology; 90.0% of CIN3 was positive for urine HPV. The corresponding sensitivity and specificity values for vulvar sampling were 92% (95% CI = 74 to 99) and 40.5% (95% CI = 25.6 to 56.7), and those for cervical sampling were 96.2% (95% CI = 80.4 to 99.9) and 40% (95% CI = 25.7 to 55.7), respectively. HPV16 was the most common carcinogenic genotype detectable in 25% of urine, 33.8% of vulvar, and 31.9% of cervical samples overall, with prevalence increasing with cervical disease grade, regardless of the sampling method. Stronger cervical HPV PCR signal strengths were associated with increased frequency of urine HPV detection. In summary, the relatively lower detection rates but comparable clinical performance of urine-based HPV sampling underscore the need for larger studies to evaluate urine-based sampling for cervical cancer screening, epidemiologic studies, and postvaccination HPV disease surveillance.

Symptotic detection of chimerism

Microchimerism (MC), transplacental acquisition of allogeneic cells from the mother (maternofetal MC) or from the fetus (fetomaternal MC) has been in the focus of research recently. Amplicons using Y-chromosome specific SRY and DYS14 sequences have been used as markers to trace cells from a male fetus in the mother. The sensitivity of these markers in formaldehyde fixed paraffin embedded samples, however, is less than optimal. To study chimerism in breast cancer we took advantage of the evolutionary history of the Y chromosome and designed amplicons on gene repeats to generate additive PCR signals. The increased sensitivity detected high incidence of male chimerism in normal breast tissues. We also showed correlation with protection from cancer with unique quantitative biology. Accumulating data from biology and medicine indicate that natural chimerism is astonishingly frequent and may affect human conditions. We hypothesize that it has significant evolutionary ramifications as well.

Combination of a modified block PCR and endonuclease IV-based signal amplification system for ultra-sensitive detection of low-abundance point mutations

By combination of a modified block PCR and endonuclease IV-based signal amplification system, we have developed a novel approach for ultra-sensitive detection of point mutations. The method can effectively identify mutant target sequence immersed in a large background of wild-type sequences with abundance down to 0.03% (for C→A) and 0.005% (for C→G). This sensitivity is among the highest in comparison with other existing approaches and the operating procedures are simple and time saving. The method holds great potential for future application in clinical diagnosis and biomedical research.

A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment

BackgroundFew studies have used quantitative polymerase chain reaction (qPCR) as an approach to measure virus neutralization assay endpoints. Its lack of use may not be surprising considering that sample nucleic acid extraction and purification can be expensive, labor-intensive, and rate-limiting.MethodsVirus/antibody mixtures were incubated for one hour at 37°C and then transferred to Vero cell monolayers in a 96-well plate format. At 24 (or 48) hours post-infection, we used a commercially available reagent to prepare cell lysates amenable to direct analysis by one-step SYBR Green quantitative reverse transcription PCR using primers specific for the RSV-N gene, thereby obviating the need for cumbersome RNA extraction and purification. The neutralization titer was defined as the reciprocal of the highest dilution needed to inhibit the PCR signal by 90% when compared with the mean value observed in virus control wells in the absence of neutralizing antibodies.ResultsWe have developed a qPCR-based neutralization assay for human respiratory syncytial virus. Due to the sensitivity of qPCR in detecting virus replication, endpoints may be assessed as early as 24 hours post-infection. In addition, the dynamic range of qPCR provides a basis for the assay to be relatively robust to perturbations in input virus dose (i.e., the assay is in compliance with the Percentage Law).ConclusionsThis qPCR-based neutralization assay is suitable for automated high-throughput applications. In addition, our experimental approach may be generalizable for the rapid development of neutralization assays for other virus families.

Insertion sequenceAS5(ISAS5) is involved in the genomic plasticity ofAeromonas salmonicida

The genome of the fish pathogen Aeromonas salmonicida subsp salmonicida harbors a large number of insertion sequences (ISs), many of which are located on plasmids. In the present study, we analyzed the small plasmid profile of A. salmonicida strains to identify evidences of plasmid alterations. Ten out of 78 strains analyzed displayed an unconventional plasmid profile. However the HER1104 strain was unique, having a positive PCR signal for pAsal1 plasmid despite not carrying this plasmid. Instead, HER1104 was bearing a plasmid at higher molecular weight than pAsal1. We characterized this new larger plasmid, which we called pAsal1B since it is a derivative of pAsal1 containing one more complete IS (ISAS5) than the parental plasmid. An additional 96 bp relic of ISAS5 was also present in pAsal1B. These results propose that ISAS5 is another active mobile genetic element in A. salmonicida subsp salmonicida and provided further proof of the genomic plasticity of this bacterium.

Challenges in Detecting HIV Persistence during Potentially Curative Interventions: A Study of the Berlin Patient

There is intense interest in developing curative interventions for HIV. How such a cure will be quantified and defined is not known. We applied a series of measurements of HIV persistence to the study of an HIV-infected adult who has exhibited evidence of cure after allogeneic hematopoietic stem cell transplant from a homozygous CCR5Δ32 donor. Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV DNA or RNA was detected in peripheral blood mononuclear cells (PBMC), spinal fluid, lymph node, or terminal ileum, and no replication-competent virus could be cultured from PBMCs. However, HIV RNA was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in ART-suppressed patients. It was not possible to obtain sequence data from plasma or gut, while an X4 sequence from PBMC did not match the pre-transplant sequence. HIV antibody levels were readily detectable but declined over time; T cell responses were largely absent. The occasional, low-level PCR signals raise the possibility that some HIV nucleic acid might persist, although they could also be false positives. Since HIV levels in well-treated individuals are near the limits of detection of current assays, more sensitive assays need to be developed and validated. The absence of recrudescent HIV replication and waning HIV-specific immune responses five years after withdrawal of treatment provide proof of a clinical cure.

An undetectable polymerase chain reaction signal in routine HIV plasma viral load monitoring is associated with better virological outcomes in patients receiving highly active antiretroviral therapy

The aim of the study was to assess whether patients with undetectable viraemia [a negative polymerase chain reaction result (PCR(neg) )] and those with plasma viral load (PVL) < 40 HIV-1 RNA copies/mL but a detectable (positive) PCR signal (PCR(pos) ) had different outcomes in terms of the development of blips and virological failure (VF). A multicentre observational database analysis was carried out. Data for patients whose highly active antiretroviral therapy (HAART) regime had been unchanged for ≥ 6 months by 1 January 2008, whose first two PVL measurements of 2008 were < 40 copies/mL and who had at least five PVL measurements between 1 January 2008 and 31 December 2010 were extracted from a multicentre observational database of 4928 patients receiving HAART. PVL assays used during this period had a detection threshold of 20 or 40 copies/mL. Undetectable PVL at baseline (BL PCR(neg) ) was defined as PCR(neg) at the first two PVL determinations of 2008. Multivariable Cox regression analysis was performed to investigate factors associated with the occurrence of blips and VF, defined as two consecutive PVL measurements > 40 copies/mL. Of the 1957 patients included in the study (mean age 47 years; median antiretroviral exposure 10.3 years), 1312 had BL PCR(neg) . Outcome events included 322 blips and 139 VFs, with incidence rates being significantly lower in patients with BL PCR(neg) than in those with BL PCR(pos) [13.0% vs. 23.4% (P < 0.0001) and 5.1% vs. 11.2% (P < 0.0001), respectively]. In multivariable analysis, BL PCR(neg) was associated with a reduced risk of blips [hazard ratio (HR) 0.58; 95% confidence interval (CI) 0.47-0.73; P < 0.0001] and VF (HR 0.44; 95% CI 0.31-0.62; P < 0.0001). Patients with PCR(neg) had better virological outcomes than those with PVL < 40 copies/mL but detectable viraemia. This suggests that the 'no-signal' information provided by currently commercially available HIV RNA quantification assays should be used routinely.

Abstract 61: Use of two-color fluorometric real-time PCR to develop molecular diagnostic tests with intrinsic quality control that augment cytomorphologic diagnosis of lung cancer.

Abstract Quantitative RT-PCR tests that measure transcript abundance of selected genes in clinical specimens promise to improve cancer diagnostic accuracy and enable “personalized medicine” through selection of the most effective treatment for each cancer. However, most clinical samples are Formalin-Fixed, Paraffin-Embedded (FFPE) and these yield sub-optimal RNA integrity. Thus, there is a need for qPCR tests with increased robustness that also have intrinsic quality control and low cost. To address this need, we developed qPCR methods that enable simultaneous measurement of each target gene and reference gene transcript relative to a known number of gene-specific internal standard (IS) molecules using two-color fluorometric analysis on real-time platform. For each gene, a competitive template IS was synthesized containing 4-6 nucleotide changes relative to the native template (NT). During PCR, NT or IS signal was quantified with sequence-specific FAM-labeled probe or Quasar 670-labeled probe, respectively. To optimize measurement sensitivity for analysis of highly degraded FFPE samples; a) each RNA sample was reverse transcribed (RT) with gene-specific primer and b) cDNA was pre-amplified with 18 PCR cycles in the presence of internal standards. Results obtained with pre-amp or no pre-amp conditions were highly correspondent. We conducted a validation study of this assay in 38 samples (10 malignant and 10 benign surgical FFPE tissues and 13 malignant and 5 benign fine needle aspirate (FNA) samples. Consistent with previous results in fresh frozen surgical samples, the lung cancer diagnostic test (LCDT) optimal cut-off value discriminated malignant from non-malignant tissues (p = 0.0009), had 92.9% specificity and 75.0% sensitivity, and a receiver operator characteristic area under the curve of 0.87 (95% confidence interval 0.74-0.99). Based on these data, we expect that this quality-controlled two color fluorometric qPCR approach will enable reliable analysis of the LCDT and other promising qPCR-based molecular diagnostic tests in small degraded RNA extracted from clinical FFPE and FNA cell block FFPE samples. Citation Format: Jiyoun Yeo, Erin L. Crawford, Thomas M. Blomquist, Lauren M. Stanoszek, Rachel E. Dannemiller, Laura Jordan, Jill Zyrek, Luis de la Casas, James C. Willey. Use of two-color fluorometric real-time PCR to develop molecular diagnostic tests with intrinsic quality control that augment cytomorphologic diagnosis of lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 61. doi:10.1158/1538-7445.AM2013-61

Combining ethidium monoazide treatment with real-time PCR selectively quantifies viable Batrachochytrium dendrobatidis cells

Detection of the lethal amphibian fungus Batrachochytrium dendrobatidis relies on PCR-based techniques. Although highly accurate and sensitive, these methods fail to distinguish between viable and dead cells. In this study a novel approach combining the DNA intercalating dye ethidium monoazide (EMA) and real-time PCR is presented that allows quantification of viable B. dendrobatidis cells without the need for culturing. The developed method is able to suppress real-time PCR signals of heat-killed B. dendrobatidis zoospores by 99.9 % and is able to discriminate viable from heat-killed B. dendrobatidis zoospores in mixed samples. Furthermore, the novel approach was applied to assess the antifungal activity of the veterinary antiseptic F10® Antiseptic Solution. This disinfectant killed B. dendrobatidis zoospores effectively within 1 min at concentrations as low as 1:6400.

Detection and quantification of a mycorrhization helper bacterium and a mycorrhizal fungus in plant-soil microcosms at different levels of complexity.

BackgroundHost plant roots, mycorrhizal mycelium and microbes are important and potentially interacting factors shaping the performance of mycorrhization helper bacteria (MHB). We investigated the impact of a soil microbial community on the interaction between the extraradical mycelium of the ectomycorrhizal fungus Piloderma croceum and the MHB Streptomyces sp. AcH 505 in both the presence and the absence of pedunculate oak microcuttings.ResultsSpecific primers were designed to target the internal transcribed spacer of the rDNA and an intergenic region between two protein encoding genes of P. croceum and the intergenic region between the gyrA and gyrB genes of AcH 505. These primers were used to perform real-time PCR with DNA extracted from soil samples. With a sensitivity of 10 genome copies and a linear range of 6 orders of magnitude, these real-time PCR assays enabled the quantification of purified DNA from P. croceum and AcH 505, respectively. In soil microcosms, the fungal PCR signal was not affected by AcH 505 in the absence of the host plant. However, the fungal signal became weaker in the presence of the plant. This decrease was only observed in microbial filtrate amended microcosms. In contrast, the PCR signal of AcH 505 increased in the presence of P. croceum. The increase was not significant in sterile microcosms that contained plant roots.ConclusionsReal-time quantitative PCR assays provide a method for directly detecting and quantifying MHB and mycorrhizal fungi in plant microcosms. Our study indicates that the presence of microorganisms and plant roots can both affect the nature of MHB-fungus interactions, and that mycorrhizal fungi may enhance MHB growth.

Molecular identification and characterization of clustered regularly interspaced short palindromic repeat (CRISPR) gene cluster in Taylorella equigenitalis

Clustered regularly interspaced short palindromic repeats (CRISPRs), of approximately 10,000 base pairs (bp) in length, were shown to occur in the Japanese Taylorella equigenitalis strain, EQ59. The locus was composed of the putative CRISPRs-associated with 5 (cas5), RAMP csd1, csd2, recB, cas1, a leader region, 13 CRISPR consensus sequence repeats (each 32 bp; 5'-TCAGCCACGTTCGCGTGGCTGTGTGTTTAAAG-3'). These were in turn separated by 12 non repetitive unique spacer regions of similar length. In addition, a leader region, a transposase/IS protein, a leader region, and cas3 were also seen. All seven putative open reading frames carry their ribosome binding sites. Promoter consensus sequences at the -35 and -10 regions and putative intrinsic ρ-independent transcription terminator regions also occurred. A possible long overlap of 170 bp in length occurred between the recB and cas1 loci. Positive reverse transcription PCR signals of cas5, RAMP csd1, csd2-recB/cas1, and cas3 were generated. A putative secondary structure of the CRISPR consensus repeats was constructed. Following this, CRISPR results of the T. equigenitalis EQ59 isolate were subsequently compared with those from the Taylorella asinigenitalis MCE3 isolate.

The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests

Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety assurance. The Biocontrol GDS® and DuPont Qualicon BAX®-RT rapid detection systems are two commercial tests based on real-time PCR amplification with potential applications for quantification of specific E. coli O157:H7 gene targets in enriched meat samples. However, there are arguments surrounding the use of these tests to predict pre-enrichment concentrations of E. coli O157:H7, as well as arguments pertaining to the influence of non-viable cells causing false positive results. The present study attempts to illustrate the effects of different bacterial physiologic states and the presence of non-viable cells on the ability of these systems to accurately measure contamination levels of E. coli O157:H7 in ground beef. While the PCR threshold cycle (CT) values of these assays showed a direct correlation with the number of bacteria present in pure cultures, this was not the case for ground beef samples spiked with various levels of injured or healthy cells. Furthermore, comparison of post-enrichment cell densities of bacteria did not correlate with injured or healthy cell numbers inoculated before enrichment process. Ground beef samples spiked with injured or healthy cells at different doses could not be distinguished by CT values from either assay. In addition, the contribution of nonviable cells in generating positive real-time PCR signals was investigated using both assays on pre-enriched and post-enriched beef samples, but only if inoculated at levels of 106 cells/sample or higher, which are levels not typically seen in ground beef.

Is Helicobacter pylori resident or transient in the human oral cavity?

Helicobacter pylori colonizes the stomachs of at least half of the world's human population. The role of the oral cavity in this colonization is not clear and there are, to date, no comprehensive data that clearly demonstrate the isolation of this bacterium from the oral cavity. The aim of this study was to evaluate the prevalence of H. pylori in the oral cavity of 15 patients who tested positive for H. pylori. A comprehensive dental examination of all patients was conducted. Samples were taken from supragingival and subgingival plaque, saliva, periapical exudates and tongue swabs. All samples were taken before the application of antibiotics. A total of 163 oral samples were investigated by PCR using two different H. pylori-specific primer pairs. A PCR inhibition control using a modified plasmid was always included for the most specific primer pair. In addition, a culture technique was used to confirm PCR results. Despite a PCR detection limit of 10(2) bacteria ml(-1), out of 14 patients, H. pylori could not be detected in any of the samples taken. In one patient, H. pylori-positive PCR signals were obtained in two samples using only one primer pair. H. pylori could not be cultivated from these two PCR-positive samples; therefore, no correlation to oral colonization status could be established. This study challenges the misleading preconception that H. pylori resides in the human oral cavity and suggests that this bacterium should be considered transient and independent of the oral status. To date, positive PCR results for H. pylori in the oral cavity have been overestimated and not critically interpreted in literature.

Aptamer-based polymerase chain reaction for ultrasensitive cell detection

A new system was developed for sensitive and selective detection of tumor cells taking advantage of cell-attached aptamers amplified by PCR and output signals amplified by cationic conjugated polymers.

The use of the Illumina FFPE Restoration Protocol to obtain suitable quality DNA for SNP-based CGH– a pilot study

The use of formalin fixed paraffin embedded (FFPE) tissues for DNA analysis has been met with great difficulties due to the degradative effect this fixation, processing and storage has on DNA quality. The lack of a suitable protocol to enhance the quality of such degraded DNA has been a great hindrance to our ability to make full use of clinically annotated FFPE cancer tissue banks. In this report we have begun to investigate the effectiveness and limitations to Illumina’s recent platform for the restoration of DNA derived from archival FFPE tissues. With the exception of select fresh frozen and blood samples, all DNA samples were extracted from FFPE tissues and restored according to Illumina’s protocol. The quality of the FFPE extracted DNA was then assessed by Illumina’s PCR-based quality control assay (QC-PCR) and the resultant DNA was subsequently run on Illumina’s SNP-based CGH chips. Chip call rates were largely used in order to determine the quality of a particular array. Overall, the chip data were highly reproducible as determined by comparing several technical replicate samples. FFPE-extracted and restored DNA performed well in comparison to DNA extracted from fresh frozen tumor and blood from the same patients, meeting the minimum standard for continuation of this platform. It should also be noted that the quality of the chip data was not appreciably enhanced by the use of sodium thiocyanate during the extraction of DNA from FFPE tissues. Moreover, chip quality was significantly lower, with regards to call rates and ‘quality’ of b allele frequency plots, when the recommended Roche DNA extraction kit was used instead of the Qiagen DNA extraction kit. Of great importance, we found that the QC-PCR provided an accurate prediction of chip quality as determined by comparing chip call rates with PCR signals derived from proprietary primer sets (rho= 0.6242, p<0.0001). The selection of restored DNA for future studies will be guided by the results from the QC-PCR assay. These preliminary data demonstrate the promise of Illumina’s DNA restoration protocol for FFPE extracted DNA from tissues older than three decades. Further studies are required to determine the full potential of this method for SNP-based CGH analysis of large FFPE tumor banks.

Absence of ectopic epithelial inclusions in 3,904 axillary lymph nodes examined in sentinel technique

Intraoperative examination of sentinel axillary lymph nodes can be done by imprint cytology, frozen section, or, most recently, by PCR-based amplification of a cytokeratin signal. Using this technique, benign epithelial inclusions, representing mammary tissue displaced along the milk line, will likely generate a positive PCR signal and lead to a false-positive diagnosis of metastatic disease. To better appreciate the incidence of ectopic epithelial inclusions in axillary lymph nodes, we have performed an autopsy study, examining on 100 μm step sections 3,904 lymph nodes obtained from 160 axillary dissections in 80 patients. The median number of lymph nodes per axilla was 23 (15, 6, and 1 in levels 1, 2, and 3, respectively). A total of 30,450 hematoxylin-eosin stained slides were examined, as well as 8,825 slides immunostained with pan-cytokeratin antibodies. Despite this meticulous work-up, not a single epithelial inclusion was found in this study, suggesting that the incidence of such inclusions is much lower than the assumed 5% reported in the literature.

HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations

AbstractMicrochimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele–specific and Y-chromosome–directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.

A simple and efficient method for DNA extraction from skin and paraffin‐embedded tissues applicable to T‐cell clonality assays

PCR-based clonality assay of rearranged T-cell receptor genes gamma and beta (TCRG and TCRB) in a number of cases could be essential to discriminate between cutaneous T-cell lymphomas and reactive lymphoproliferative lesions in the skin. However, extraction of good-quality DNA from skin specimens (especially formalin-fixed paraffin-embedded) remains a challenge. Common procedures, being labour-intensive and time-consuming and requiring toxic solvents such as phenol and chloroform, still may end up with DNA sample of insufficient quality. We herewith present a simple and efficient method for DNA isolation based on ammonia extraction of tissue, followed by neutralization and simultaneous salting out of proteins with acetic acid. We have analysed 30 samples - 24 fresh (16 skin, two spleen and six lymph node) and six paraffin-embedded. Standard procedure (proteinase K digestion, followed by phenol/chloroform extraction) has been carried out simultaneously. We observed good PCR signal for TCRG rearrangements in 30 samples processed with the new protocol and only in 20 extracted with proteinase K/phenol/chloroform. For TCRB, the success rate was 29 of 30 with the new protocol, compared to 11 of 30 with conventional protocol. The proposed method of DNA extraction should improve the value of T-cell clonality assay, because insufficient DNA quality and quantity may bias analysis towards monoclonality and therefore cause false-positive results.