The current study aimed to test the validity of the Implicit Relational Assessment Procedure (IRAP), as compared to the Implicit Association Test (IAT), by assessing the attitudes of Dublin dwellers and rural dwellers towardThe currently most popular and well-researched measure of so-called implicit attitudes is the Implicit Association Test, or IAT (Greenwald, McGhee, & Schwartz, 1998). The core assumption underpinning the method is that individuals should respond quickly when asked to emit a similar response for two concepts that are closely associated in memory, but should respond more slowly when the two concepts are less closely associated. The first IAT study by Greenwald et al. (1998) asked participants in one task to categorize the names of flowers with positive words and the names of insects with negative words, but in a second task these categorizations were reversed (flowers-negative and insects-positive). The predicted IAT effect was observed: The participants responded faster on flower-positive andWe would like to thank two anonymous reviewers for their thoughtful and constructive reviews of the original manuscript.
Objective The T-cell receptor β constant region 1 (TRBC1) antibody can identify T-cell clonality and distinguish pathological from normal T cells. This study aims to establish optimal cutpoints for establishing monotypia and validate the diagnostic abilities of the TRBC1 antibody when used as a reflex test in conjunction with an existing T-cell antibody panel. Materials and Methods We used 46 normal peripheral blood specimens and examined 8 patients with reactive lymphoproliferations to determine the normal biological range of TRBC1 on CD4+ and CD8+ T cells. We also evaluated 43 patient specimens that were submitted for investigation of a lymphoproliferative disorder for CD2/CD3/CD4/CD5/CD7/CD8/CD16/CD26/CD45/CD56/TCR αβ/TCR γδ, along with TRBC1 expression. The results were compared to TCR gene rearrangement patterns using polymerase chain reaction (PCR) analysis. Results Statistical analysis established differing cutoff points for establishing monotypia dependent on restricted TRBC1 or TRBC2 usage. Direct comparison with molecular analysis indicated that no specimen identified with the restricted expression of TRBC1 was reported as polyclonal by PCR with a concordance rate of 97% between a clonal PCR result and monotypic TRBC1 expression. Conclusion Incorporation of the TRBC1 antibody using statistically derived cutoff points in a reflex setting for the evaluation of a suspected T-cell neoplasm improves the identification of clonal T-cell populations by flow cytometry and correlates well with molecular methods.
Complications of Gastrooesophageaalreflux disease (GORD) are associated with supine or combined reflux. Upright reflux has been reported to be of less severity, but some reports have suggested a correlation between oesophagitis and post-prandial reflux (PPR).200 patients with reflux symptoms were analysed to establish the relative frequencies of uptight, supine and combined rellux, Further analysis was then carried out to establish the contribution of PPR. PPR was defined as % pH < 4 during periods of 60 minutes after eating, Results: 144 patients had abnormal pH profile, significant upright reflux being detected in 111 (77. I%), This was combined with significant supine reflux in 74 (66.6%) of the above. 37 (25.7%) of the total abnormal population had upright reflux alone and 33 (22.9%) had supine reflux only. In the pure upright refluxers (group A) PPR was < pH 4.0 for a mean of 17.1% of the total post-prandial time, and accounted for 30.2% of the total reflux time. In combined retluxers (group B) the mean duration of PPR was 27.6% of the post prandial time and accounted for 23.4% of total reflux. Supine refluxers (group C) PPR duration was 5.2% which represented 13% of total reflux in this group.Total % PPR % Restored to Normal Group A (n=37) 9.5 (7.5) 17,1(16.3) 51.0"** Group B (n = 74) 22.5 (16.6) 27.6 (21,1) 8.1% Group C (n = 33) 10.9 (8.3) 5.2 (4.2) 0%Mean % (+SD) *** p < 0.00I, Chi-square test.Elimination of PPR by subtracting PPR time from total reflux time restored 19 (51%) of group A and 6 (8.1%) of group B patients to within normal range, It is concluded that PPR is a significant factor in upright reflux which has implications for treatment. 2HOW GOOD IS OPEN COMMON BILE DUCT EXPLORATION?
Genetic makers such as TP53, ATM mutations and IgH somatic hypermutation status have been used to predict clinical behaviour and response to therapy in Chronic Lymphocytic Leukaemia (CLL) for the last decade. Recently identified genetic markers including splicing factor 3 B1 (SF3B1), NOTCH and BirC3 are also significant but their role cannot be fully evaluated until a robust assay is available for a routine molecular diagnostic laboratory The cell spliceosome is involved in the precise excision of introns from pre-mRNA and is composed of five subunits of which SF3B1 is a core component. Patient samples with SF3B1 mutations display enhanced intron retention in a small subset of transcripts involved in cancer related regulation such as cell cycle control, apoptosis and angiogenesis. Studies of CLL patient samples has shown that those with mutations in SF3B1 have been associated with shorter treatment free and overall survival. The screening methodology for SF3B1 mutations through deep sequencing, though very sensitive, is not available in routine diagnostic laboratories. Approximately 90% of CLL-associated SF3B1 mutations occur is 336bp region of the cDNA exome amenable to PCR amplification. We designed primers flanking this region and following reverse transcription (RT) PCR amplification, detected the products on standard agarose gels. Following bidirectional Sanger sequencing, we compared the sequences obtained with reference SF3B1 cDNA/amino acid code. We assessed the impact of the mutations by the polyphen database and compared this to the immunophenotype and cytogenetic data as well as response to treatment through minimal residual disease (MRD) tracking. We subsequently designed an assay for detection of sequence variants in the SF3B1 region of interest by High Resolution Melt (HRM) curve analysis. This has enabled us to identify genetic variants of the SF3B1 region prior to sequencing. The application of this HRM pre-sequencing screening assay has resulted in a significant reduction in the number of samples requiring sequencing. The patient cohort consisted of 52 patients enrolled on a Phase II, multi centre study of fludarabine, cyclophosphamide and rituximab (FCR) and included patients < 65 yrs, WHO status of 0, 1 or 2 without a deletion 17p. All samples were collected and tested prior to treatment, at the end of treatment (after 4 or 6 cycles when MRD negative), 6 monthly post treatment for 5 years for minimal residual disease and then annually by 6 colour immunophenotyping. Patients (51/52, no RNA available for 1 patient) were screened prior to treatment for the presence of SF3B1 mutations. Five of 51 (9.8 %) patients had SF3B1 mutations detectable by this methodology shown in the table below. Table 1. Pt. Nucleotide change Amino acid change Polyphen IGHV M=mutatedU=unmutated Cytogenetics Immunophenotyping MRD 1 c.2146A>G p.K700E Damaging U V4-6*01 Normal CD5 -ve Not tracked due to neg CD5 2 c.2179G>C p.A711P Damaging U V1-69*01 Normal Neg @ 3 mts Pos @ 16 mts 3 c.2267G>A p.G740E Damaging M V3-74*01 del 13q CD5 weak +ve CD38 +ve Neg @ 3 mts Pos @ 12 mts 4 c.2146A>G p.K700E Damaging U V1-69*01 del 13q Never MRD neg 5 c.2032C>T p.H662Y Damaging U V3-11*01 del 13q CD38 +ve Withdrawn Patients with mutations in SF3B1 all had favourable cytogenetics (normal or del 13q) and the majority (4/5) had unmutated IGHV genes. We detected two patients with a mutation at c.2146A>G, p.K700E and one at c.2267G>A, pG740E which have been described previously and two novel mutations at c.2179G>C, p.A711P and c.2032C>T, p.H662Y. All mutations detected were damaging as per the polyphen database. Two patients have weak or no expression of CD5. One patient has never achieved MRD negativity, and two patients who became MRD negative at 3 months reverted to MRD positivity 9 and 13 months later respectively. One patient's MRD levels could not be tracked due to negative CD5 expression and was followed by a less sensitive IgH clonality assay (sensitivity approx. 1%) and one patient was withdrawn from the study. In summary, we have developed a simple PCR based technique to detect SF3B1 mutations in CLL patient samples with high disease burden which can be applied in routine molecular diagnostic laboratories without access to next generation sequencing. The use of HRM technology resulted in a > 90% reduction in the number of samples requiring sequencing. Disclosures No relevant conflicts of interest to declare.
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