Allergen-specific serum immunoglobulin E (sIgE) levels are a cornerstone in allergy diagnostics. While immunoglobulins are known to be relatively stable molecules, the influence of preanalytical factors on the stability of sIgE is not thoroughly investigated. We studied the effect of several preanalytical factors: (1) delayed centrifugation of serum samples in tubes with separating gel for 10, 24 and 48 h, (2) prolonged storage at 5 ? for 3, 7, 10 and 14 days, (3) storage tube type (primary tube with separating gel or secondary tube), (4) repeated freeze-thawing cycles, and (5) prolonged storage at -20 ? for 4 and 8 weeks. We found that sIgE is stable at room temperature for 48 h before centrifugation and for 10 days at 5 ? after centrifugation. There was no effect of the separating gel after storing serum for 1 week in the freezer. However, storage for 4-8 weeks, and introducing more than one freeze-thaw cycle resulted in a larger variation of sIgE levels. In conclusion, we found that sIgEs are stable under various preanalytical conditions, which allows for flexible handling of samples for a comprehensive portfolio of sIgE analyses.

INTRODUCTION: In Denmark, diagnosing and treating allergy is mainly performed by general practitioners (GPs), but precise expectations of the GPs are not described in guidelines. Furthermore, very little is known about GPs' use of allergen-specific immunoglobulin E (sIgE) tests. The aim of this study was to describe the use of these tests in the Central Denmark Region. METHODS: We performed analyses on data from all sIgE tests ordered by GPs in the Central Denmark Region in 2015. A test was considered positive if the serum level of IgE was >= 0.35 kU/l. RESULTS: Serum levels of sIgE were determined in 26,129 patients, equivalent to 2% of the Danish population. A total of 106,237 tests were performed, the majority as part of screening algorithms for inhalant and food allergens. Screening was ordered 20,697 times for inhalation allergens and 12,999 times for food allergens. Additionally, a considerable number of tests for antibiotics (n = 4,407), insect venom (n = 748) and other allergens were performed (n = 824). Positive rates were determined for various allergens in relation to gender and age. The rates were generally higher than rates known to be present in the background population. A higher percentage of females than males was tested. However, positive rates were generally lower in females than in males. CONCLUSIONS: This is the first descriptive analysis of the use of testing for sIgE in general practice. Results from this study may be used to optimise how GPs order and interpret sIgE tests in the future.

Objectives During a comprehensive patient safety program at a 550-bed regional hospital in the Capital Region of Denmark, we observed an unexpected and unexplained doubling of the median patient harm rate from 56 to 109 harms per 1000 patient days measured by the Institute for Healthcare Improvement Global Trigger Tool (GTT). Meanwhile, other measures of patient safety, including hospital standardized mortality ratio, were stable or improving. Moreover, the review team was very experienced and stable during this period. Thus, we hypothesized that the increase in harm rate was not a true reflection of increased risk of patient harm but the result of the team getting better at identifying harms during GTT reviews. Methods We examined the ability of the GTT review team to reproduce the rate of harm of two separate periods in the same hospital: period 1 (January-June 2010) and period 2 (October 2011-March 2012). For each period, we examined two samples: the original sample that was drawn and used for the ongoing monitoring of harm at the hospital during the safety campaign and a second that we drew and analyzed for this study. Results We found increased harm rates both between review 1 and review 2 and between period 1 and period 2. The increase was solely in category E, minor temporary harm. Conclusions The very experienced GTT team could not reproduce harm rates found in earlier reviews. We conclude that GTT in its present form is not a reliable measure of harm rate over time.

INTRODUCTION:Chronic inflammatory bowel disease (IBD) is characterized by recurrent inflammation of the intestinal mucosa. Reactive molecules play a central role in altering the intestinal permeability, which may induce or sustain an immune response. Changes in detoxification of substances that causes epithelial damage may confer susceptibility to IBD. Hence, polymorphic enzymes involved in the detoxification processes may be risk factors of IBD.METHODS:The two biotransformation enzymes microsomal epoxide hydrolase and N-acetyltransferase 2 were genotyped using TaqMan based real-time PCR in 388 patients with Crohn's disease, 565 patients with ulcerative colitis and 796 healthy controls.RESULTS:No association was found between the genotypes of low microsomal epoxide hydrolase activity or slow N-acetyltransferase 2 acetylator status and IBD. An association was found between microsomal epoxide hydrolase and less than 40 years of age at diagnosis of Crohn's disease and microsomal epoxide hydrolase and azathiporine use in patients with ulcerative colitis. No other evident phenotypic associations were found for the two enzymes and either ulcerative colitis or Crohn's disease. A possible modification of smoking on microsomal epoxide hydrolase genotypes was found.CONCLUSION:Microsomal epoxide hydrolase and N-acetyltransferase 2 genotypes appear not to be individual risk factors of IBD, or to be important in relation to phenotypic characteristics of IBD.

Early relapse detection in acute myeloid leukemia is possible using standardized real-time quantitative polymerase chain reaction (RQ-PCR) protocols. However, optimal sampling intervals have not been defined and are likely to vary according to the underlying molecular lesion. In 74 patients experiencing hematologic relapse and harboring aberrations amenable to RQ-PCR (mutated NPM1 [designated NPM1c], PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11), we observed strikingly different relapse kinetics. The median doubling time of the CBFB-MYH11 leukemic clone was significantly longer (36 days) than that of clones harboring other markers (RUNX1-RUNX1T1, 14 days; PML-RARA, 12 days; and NPM1c, 11 days; P < .001). Furthermore, we used a mathematical model to determine frequency of relapse detection and median time from detection of minimal residual disease to hematologic relapse as a function of sampling interval length. For example, to obtain a relapse detection fraction of 90% and a median time of 60 days, blood sampling every sixth month should be performed for CBFB-MYH11 leukemias. By contrast, in NPM1c(+)/FLT3-ITD(-), NPM1c(+)/FLT3-ITD(+), RUNX1-RUNX1T1, and PML-RARA leukemias, bone marrow sampling is necessary every sixth, fourth, and fourth and second month, respectively. These data carry important implications for the development of optimal RQ-PCR monitoring schedules suitable for evaluation of minimal residual disease-directed therapies in future clinical trials.

Introduction. A combination of genetic predisposition and interactions with environmental factors are believed to be responsible for disease phenotype and disease progression in inflammatory bowel diseases. The harmful effect of smoking and other environmental factors is believed to be highly dependent on the activity of detoxification enzymes. The aims of the study were to examine possible associations between the detoxifying glutathione S-transferases (GSTs) family mu, theta and pi gene variants and inflammatory bowel disease, and secondly to examine a potential genotype genotype interaction between these variants. Genotype disease phenotype associations and a possible interaction between genotype and cigarette smoking were also assessed. Methods. Three hundred and eighty-eight patients with Crohn's disease (CD), 565 patients with ulcerative colitis (UC) and 796 healthy Danish controls were included in the study. Genomic DNA was used for genotyping of the GST genes using PCR or real-time PCR. Results. No associations were found between GST genotypes and inflammatory bowel diseases. Neither did a combination of the GST genotypes reveal any associations. No genotype disease phenotype associations were found. Smoking was positively associated with CD and negatively associated with UC. An interaction between smoking and GSTM1*0 genotype was found for UC, where the GSTM1*0 genotype appear to strengthen the protective effect of smoking on disease susceptibility. Conclusion. The GST genotypes do not seem to be important in susceptibility of inflammatory bowel disease in the Danish population. Nor did we find convincing evidence of associations between GST genotype and phenotypic features of inflammatory bowel diseases.

BACKGROUND:Crohn's disease (CD) and ulcerative colitis (UC) are characterized by a dysregulated inflammatory response to normal constituents of the intestinal flora in the genetically predisposed host. Heme oxygenase-1 (HO-1/HMOX1) is a powerful anti-inflammatory and anti-oxidant enzyme, whereas the pro-inflammatory interleukin 1 beta (IL-1 beta/IL1B) and anti-inflammatory interleukin 10 (IL-10/IL10) are key modulators for the initiation and maintenance of inflammation. We investigated whether single nucleotide polymorphisms (SNPs) in the IL-1 beta, IL-10, and HO-1 genes, together with smoking, were associated with risk of CD and UC.METHODS:Allele frequencies of the IL-1 beta T-31C (rs1143627), and IL-10 rs3024505, G-1082A (rs1800896), C-819T (rs1800871), and C-592A (rs1800872) and HO-1 A-413T (rs2071746) SNPs were assessed using a case-control design in a Danish cohort of 336 CD and 498 UC patients and 779 healthy controls. Odds ratio (OR) and 95% confidence interval (95% CI) were estimated by logistic regression models.RESULTS:Carriers of rs3024505, a marker polymorphism flanking the IL-10 gene, were at increased risk of CD (OR = 1.40, 95% CI: 1.06-1.85, P = 0.02) and UC (OR = 1.43, 95% CI: 1.12-1.82, P = 0.004) and, furthermore, with risk of a diagnosis of CD and UC at young age (OR = 1.47, 95% CI: 1.10-1.96) and OR = 1.35, 95% CI: 1.04-1.76), respectively). No association was found between the IL-1 beta, IL-10 G-1082A, C-819T, C-592A, and HO-1 gene polymorphisms and CD or UC. No consistent interactions between smoking status and CD or UC genotypes were demonstrated.CONCLUSIONS:The rs3024505 marker polymorphism flanking the IL-10 gene was significantly associated with risk of UC and CD, whereas no association was found between IL-1 beta or HO-1 gene polymorphisms and risk of CD and UC in this Danish study, suggesting that IL-10, but not IL-1 beta or HO-1, has a role in IBD etiology in this population.

Objective. Crohn's disease (CD) and ulcerative colitis (UC) are characterized by an impaired mucosal defence to normal constituents of the intestinal flora and a dysregulated inflammatory response. The purpose of the study was to investigate whether single nucleotide polymorphisms (SNPs) in genes involved in these processes were associated with CD and UC. Material and methods. Allele frequencies of the cyclooxygenase 2 (COX-2/PTGS2/PGHS2) G-765C and breast cancer resistance protein (BCRP/ABCG2) C421A as well as allele and haplotype frequencies of multidrug resistance 1 (MDR1, ABCB1) SNPs G2677T/A, C3435T and G-rs3789243- A (intron 3) were assessed in a Danish case-control study comprising 373 CD and 541 UC patients and 796 healthy controls. Results. Carriers of the homozygous COX-2 and MDR1 intron 3 variant had a relatively high risk of CD, odds ratio (95% CI) (OR (95% CI)) = 2.86 ((1.34-5.88) p = 0.006) and 1.39 ((0.99-1.92) p = 0.054), respectively, and for UC of 2.63 ((1.33-5.26) p = 0.005) and 1.28 ((0.96-1.51) p = 0.093), respectively, assuming complete dominance. No association was found for BCRP or other MDR1 SNPs, or for selected MDR1 haplotypes. No effect-modification of smoking habit at the time of diagnosis was found. Conclusions. An effect of the COX-2 polymorphism on both CD and UC was shown which is compatible with the presence of a recessive allele in linkage equilibrium with the SNP marker in the COX-2 gene. The polymorphism located in intron 3 of the MDR1 gene showed a weak association with CD, and a marginally suggestive association with UC.

In haematological malignancies, molecular markers like fusion DNA from balanced translocations, point mutations, or over-expressed genes can now be used not only for diagnosis, but also for determination of the minimal residual disease (MRD) after cytoreduction with a sensitivity by far exceeding that of previous methodologies. The sensitivity of quantitative polymerase chain reaction typically reaches a validated identification of 1 malignant cell in 100,000.

The translation of quantitative polymerase chain reaction technology for the quantitative detection of minimal residual disease (MRD) arising from molecular aberrations in leukaemias has progressed from the pre-clinical setting to daily clinical practice. Thus, it is now part of the mainstay in following patients with chronic myeloid leukaemia treated with the tyrosine kinase inhibitor imatinib. Moreover, the methodology is being integrated in an increasing number of clinical trials, where it is expected to result in more rational and individualized clinical decision-making. A special point in favour of the techniques is the powerful and early detection of disease relapses, in some cases up to one year prior to clinical detection.

BACKGROUND:The xenobiotic transporters, Multidrug Resistance 1 (MDR1/ABCB1) and Breast Cancer Resistance Protein (BCRP/ABCG2) may restrict intestinal absorption of various carcinogens, including heterocyclic amines (HCA) and polycyclic aromatic hydrocarbons (PAH). Cyclooxygenase-2 (COX-2) derived prostaglandins promote gastrointestinal carcinogenesis, affecting angiogenesis, apoptosis, and invasiveness.The aim of this study was to investigate if polymorphisms in these genes were associated with risk of colorectal cancer (CRC), and to investigate possible interactions with lifestyle factors such as smoking, meat consumption, and NSAID use.METHODS:The following polymorphisms were analyzed; a synonymous MDR1 C3435T (rs1045642) in exon26, G-rs3789243-A in intron3, the functional BCRP C421A (rs2231142), the two COX-2 A-1195G (rs689466) and G-765C (rs20417) in the promoter region, and the COX-2 T8473C (rs5275) polymorphisms in the 3'-untranslated region. The polymorphisms were assessed together with lifestyle factors in a nested case-cohort study of 359 cases and a random cohort sample of 765 participants from the Danish prospective Diet, Cancer and Health study.RESULTS:Carriers of the variant allele of MDR1 intron 3 polymorphism were at 1.52-fold higher risk of CRC than homozygous wild type allele carriers (Incidence rate ratio (IRR) = 1.52, 95% Confidence Interval (CI): 1.12-2.06). Carriers of the variant allele of MDR1 C3435T exon 26 had a lower risk of CRC than homozygous C-allele carriers (IRR = 0.71 (CI:0.50-1.00)). There was interaction between these MDR1 polymorphisms and intake of red and processed meat in relation to CRC risk. Homozygous MDR1 C3435T C-allele carriers were at 8% increased risk pr 25 gram meat per day (CI: 1.00-1.16) whereas variant allele carriers were not at increased risk (p for interaction = 0.02). COX-2 and BCRP polymorphisms were not associated with CRC risk. There was interaction between NSAID use and MDR1 C3435T and COX-2 T8473C (p-values for interaction 0.001 and 0.04, respectively).CONCLUSION:Two polymorphisms in MDR1 were associated with CRC risk and there was interaction between these polymorphisms and meat intake in relation to CRC risk. Our results suggest that MDR1 polymorphisms affect the relationship between meat and CRC risk.

Known genetic determinants contributing to risk of the inflammatory bowel diseases (IBD), Crohns disease (CD) and ulcerative colitis (UC), are nearly absent in the Northern European countries including the Scottish and Danish populations. Cyclo-oxygenase-2 (COX-2) plays a key role in regulation of the inflammatory response. COX-2 gene polymorphisms (SNP) have previously been associated with IBD. COX-2 haplotypes in the Scottish and Danish IBD populations were investigated. Genotype and haplotype distributions of the COX-2/PTGS2/PGHS2 A-1195G (rs689466), G-765C (rs20417), and T8473C (rs5275) polymorphisms were assessed using a case control design in a Scottish (423 CD, 510 UC and 404 healthy controls) and a Danish (309 CD, 463 UC and 703 healthy controls) cohort. Logistic regressions was used for the statistical analyses. No crude association was found between COX-2 genotypes and CD. Regarding UC, carriers of the COX-2 A-1195G variant allele had increased risk of UC as a whole and among never-smokers (odds ratio (95% confidence interval) 1.53 (1.03-2.27) and 1.82 (1.08-3.06), respectively) in the Scottish study group, but not in the Danish study group. Smoking status is a major determinant of the risk of IBD regardless of genotype. Our comprehensive case-control study did not support an important role of COX-2 in IBD development as no consistent associations were found in these two independent, but presumable genetically related, cohorts. Smoking had a strong risk-modifying effect and inclusion of smoking status is essential in the evaluation of the role of genetic predisposition in IBD.

BACKGROUND:Smoking, dietary factors, and alcohol consumption are known life style factors contributing to gastrointestinal carcinogenesis. Genetic variations in carcinogen handling may affect cancer risk. The multidrug resistance 1(MDR1/ABCB1) gene encodes the transport protein P-glycoprotein (a phase III xenobiotic transporter). P-glycoprotein is present in the intestinal mucosal lining and restricts absorption of certain carcinogens, among these polycyclic aromatic hydrocarbons. Moreover, P-glycoprotein transports various endogenous substrates such as cytokines and chemokines involved in inflammation, and may thereby affect the risk of malignity. Hence, genetic variations that modify the function of P-glycoprotein may be associated with the risk of colorectal cancer (CRC). We have previously found an association between the MDR1 intron 3 G-rs3789243-A polymorphism and the risk of CRC in a Danish study population. The aim of this study was to investigate if this MDR1 polymorphism was associated with risk of colorectal adenoma (CA) and CRC in the Norwegian population.METHODS:Using a case-control design, the association between the MDR1 intron 3 G-rs3789243-A polymorphism and the risk of colorectal carcinomas and adenomas in the Norwegian population was assessed in 167 carcinomas, 990 adenomas, and 400 controls. Genotypes were determined by allelic discrimination. Odds ratio (OR) and 95 confidence interval (95% CI) were estimated by binary logistic regression.RESULTS:No association was found between the MDR1 polymorphism (G-rs3789243-A) and colorectal adenomas or cancer. Carriers of the variant allele of MDR1 intron 3 had odds ratios (95% CI) of 0.97 (0.72-1.29) for developing adenomas, and 0.70 (0.41-1.21) for colorectal cancer, respectively, compared to homozygous wild type carriers.CONCLUSION:The MDR1 intron 3 (G-rs3789243-A) polymorphism was not associated with a risk of colorectal adenomas or carcinomas in the present Norwegian study group. Thus, this MDR1 polymorphism does not seem to play an important role in colorectal carcinogenesis in this population.

In acute myeloid leukemia (AML) mouse models, the RUNX1-RUNX1T1 fusion protein has failed to produce leukemia by itself, but alternative splicing of exon 9a of the RUNX1-RUNX1T1 fusion transcript (FT) has recently been shown to enhance the leukemogenic potential. We have analyzed 138 diagnosis and follow-up samples from 13 RUNX1-RUNX1T1+ patients as well as diagnosis samples from 13 RUNX1-RUNX1T1- AML patients and 26 healthy donors. Levels of native RUNX1T1 mRNA were low in both healthy and RUNX1-RUNX1T1-negative AML samples. Likewise, the ratio between RUNX1T1 mRNA harboring exon 9a and lacking exon 9a was low and tightly regulated (0.017-0.11). In contrast, 11/13 RUNX1-RUNX1T1-positive AML patients displayed high and variable ratios of FT ranging from 0.05 to 0.46 (P < 0.001, Wilcoxon rank-sum test), indicating altered exon 9a splicing in these patients. Importantly, patients who remained in continuous complete remission displayed a faster disappearance of the RUNX1-RUNX1T1 exon 9a splice variant compared to patients bound to relapse (P = 0.02). In conclusion, alternative splicing seems to be part of the leukemogenic process in the majority of RUNX1-RUNX1T1-positive AML patients, and splice variant kinetics under cytoreduction may be a predictor for patients prone to relapse.

INTRODUCTION: In 10% of acute myeloid leukemia (AML) patients the recurrent balanced translocation t(8;21) places the RUNX1 (AML1) gene next to RUNX1T1 (ETO) gene resulting in the generation of a RUNX1-RUNX1T1 fusion protein. While in mouse models this protein has failed to produce leukemia alone, alternative splicing of exon 9a of the RUNX1-RUNX1T1 fusion transcript has recently (Yan et al, Nat Med , 12, 945, 2006) been shown to result in a truncated RUNX1-RUNX1T1 fusion protein with increased leukemogenic potential in a retroviral transduction-transplantation model.

Multiple myeloma is associated with a high risk of infections. We hypothesized that Fc gamma receptor (FCGR) and myeloperoxidase (MPO) promoter gene polymorphisms influence the risk of infections after induction chemotherapy (IC) and autologous stem cell transplantation (ASCT). Retrospectively, we analysed 136 patient courses of IC and 113 procedures of ASCT. Genetic analyses were made with PCR techniques on genomic DNA. The incidence rate ratio of sepsis during ASCT in patients homozygous for the G-129MPO promoter type was 0.30 (95% CI: 0.09-0.96). The G-463AMPO promoter polymorphism was not associated with the risk of infections. The polymorphisms of FCGR2A, FCGR3A and FCGR3B were not convincingly associated with infections. The NA1 variant of FCGR3B was strongly skewed with other risk factors, and the results in IC and ASCT were conflicting. Further studies of the G-129AMPO promoter as a potential risk modifier for infections are relevant.

We hypothesized that Wilms tumour 1 gene (WT1) expression levels in acute myeloid leukaemia (AML) patients might have predictive value and reveal molecular relapse kinetics. WT1 level was determined at diagnosis, during therapy and post-therapy follow-up in 89 patients who reached first complete remission (CR1) (952 samples, median 8 samples/patient, range 2-38). CR1 bone marrow (BM) WT1 level above normal (based on 39 healthy donors) was an independent adverse prognostic factor regarding both disease-free survival [hazard ratio (HR) 4.46, P = 0.001] and overall survival (HR 2.62, P = 0.019). By grouping 34 BM and 99 peripheral blood (PB) complete remission samples in monthly intervals prior to clinical relapse, disease development was delineated and a simple mathematical model constructed, that allowed for the prediction of relapse detection rates (RDRs) as well as median times [t(m)s] from WT1 positivity to clinical relapse. BM sampling was required to obtain RDRs above 93% and t(m)s above 67 d. Acceptable RDRs and t(m)s (81% and 44 d, respectively) could be acquired by bimonthly PB sampling. In conclusion, CR1 WT1 expression is an independent prognostic factor in AML. According to our model, BM is superior for relapse prediction, but PB samples are useful when shorter sampling intervals are possible.