Practice of percutaneous needle autopsy; a descriptive study reporting experiences from Uganda

BACKGROUND: Percutaneous needle autopsy can overcome a number of barriers that limit the use of complete autopsies. We performed blind-and ultrasound guided needle autopsies in HIV-infected adults in Uganda. In this study we describe in detail the methods we used, the ability of both procedures to obtain sufficient tissue for further examination and the learning curve of the operators over time. METHODS: If written informed consent was granted from the next of kin, we first performed a blind needle autopsy, puncturing brain, heart, lungs, liver, spleen and kidneys using predefined surface marking points. We then performed an ultrasound guided needle autopsy puncturing heart, liver, spleen and kidneys. The number of attempts, expected success and duration of the procedure were noted. A pathologist read the slides and indicated if the target tissue was present and of sufficient quality for pathological review. We report the predicted and true success rates, compare the yield of blind to ultrasound guided needle biopsies and evaluate the failure rate over time. RESULTS: Two operators performed 96 blind needle autopsies and 95 ultrasound guided needle autopsies. For blind needle biopsies true success rates varied from 56-99% and predicted success rates from 89-99%. For ultrasound guided needle biopsies true success rates varied from 72-100% and predicted success rates from 84-98%. Ultrasound guidance led to a significantly higher success rate in heart and left kidney. A learning curve was observed over time with decreasing failure rates with increasing experience and a shorter duration of the needle autopsy. CONCLUSION: Needle autopsy can successfully obtain tissue for further pathological review in the vast majority of cases, with a decrease in failure rate with increasing experience of the operator. The benefit of ultrasound guidance will depend on the population, the disease and organ of interest and the local circumstances. Our results justify further evaluation of needle autopsies as a method to establish a cause of death

Array-Based DNA Methylation Profiling for Breast Cancer Subtype Discrimination

BACKGROUND: Abnormal DNA methylation is well established for breast cancer and contributes to its progression by silencing tumor suppressor genes. DNA methylation profiling platforms might provide an alternative approach to expression microarrays for accurate breast tumor subtyping. We sought to determine whether the distinction of the inflammatory breast cancer (IBC) phenotype from the non-IBC phenotype by transcriptomics could be sustained by methylomics. METHODOLOGY/PRINCIPAL FINDINGS: We performed methylation profiling on a cohort of IBC (N = 19) and non-IBC (N = 43) samples using the Illumina Infinium Methylation Assay. These results were correlated with gene expression profiles. Methylation values allowed separation of breast tumor samples into high and low methylation groups. This separation was significantly related to DNMT3B mRNA levels. The high methylation group was enriched for breast tumor samples from patients with distant metastasis and poor prognosis, as predicted by the 70-gene prognostic signature. Furthermore, this tumor group tended to be enriched for IBC samples (54% vs. 24%) and samples with a high genomic grade index (67% vs. 38%). A set of 16 CpG loci (14 genes) correctly classified 97% of samples into the low or high methylation group. Differentially methylated genes appeared to be mainly related to focal adhesion, cytokine-cytokine receptor interactions, Wnt signaling pathway, chemokine signaling pathways and metabolic processes. Comparison of IBC with non-IBC led to the identification of only four differentially methylated genes (TJP3, MOGAT2, NTSR2 and AGT). A significant correlation between methylation values and gene expression was shown for 4,981 of 6,605 (75%) genes. CONCLUSIONS/SIGNIFICANCE: A subset of clinical samples of breast cancer was characterized by high methylation levels, which coincided with increased DNMT3B expression. Furthermore, an association was observed with molecular signatures indicative of poor patient prognosis. The results of the current study also suggest that aberrant DNA methylation is not the main force driving the molecular biology of IBC

The therapeutic effect of the neuropeptide hormone somatostatin on Schistosoma mansoni caused liver fibrosis

BACKGROUND: The neuropeptide somatostatin is one of the major regulatory peptides in the central nervous system and the digestive tract. Our recent work has delineated an association between fibrosis and low levels of endogenous somatostatin plasma levels in Schistosoma mansoni infected subjects. Based on these results this paper explores the therapeutic potential of somatostatin in a mouse model of hepatic fibrosis associated with S. mansoni infections. METHODS: Groups of outbred Swiss mice were infected with 100 S. mansoni cercariae, infection maintained till weeks 10 or 14, and then somatostatin therapy delivered in two regimens – Either a one or a two-day treatment. All animals were sacrificed one week after therapy and controlled for liver, spleen and total body weight. Circulating somatostatin levels in mice plasma were measured at the time of sacrifice by means of a radio-immuno assay. GraphPad Prism(® )was used for statistical calculations. RESULTS: Somatostatin administration showed little toxicity, probably due to its short half-life. Total liver and spleen weights of S. mansoni infected animals increased over time, with no changes observed due to somatostatin therapy. Total body weights were decreased after infection but were not affected by somatostatin therapy. Snap frozen liver sections were stained with haematoxylin-eosin or Masson's trichrome to study parasite count, hepatocyte status, granuloma size and cellularity. After somatostatin treatment mean egg counts per liver section (43.76 ± 3.56) were significantly reduced as compared to the egg counts in untreated mice after 10 weeks of infection (56.01 ± 3.34) (P = 0.03). Similar significant reduction in parasite egg counts were also observed after somatostatin treatment at 14 weeks of infection (56.62 ± 3.02) as compared to untreated animals (69.82 ± 2.77)(P = 0.006). Fibrosis was assessed from the spectrophotometric determination of tissue hydroxyproline. Infection with S. mansoni caused increased hydroxyproline levels (9.37 ± 0.63 μmol at wk10; 9.65 ± 0.96 μmol at wk14) as compared to uninfected animals (1.06 ± 0.10 μmol). This significant increase in collagen content (P = 0.01; 0.007 respectively) marks the fibrosis observed at these time points. Treatment with somatostatin resulted in a significant decrease in hydroxyproline levels both at wk10 (4.76 ± 0.58 μmol) and at wk14 (5.8 ± 1.13 μmol) (P = 0.01; 0.03 respectively). Endogenous somatostatin levels were increased at wk10 (297 ± 37.24 pg/ml) and wk14 (206 ± 13.30 pg/ml) of infection as compared to uninfected mice (119 ± 11.99 pg/ml) (P = 0.01; 0.008 respectively). Circulating somatostatin levels in infected animals were not significantly affected by somatostatin treatment. Hepatocyte status remained unaltered and granulomas were not remarkably changed in size or cellularity. CONCLUSION: Our experiments reveal an antifibrotic effect of somatostatin in schistosomiasis. We have previously shown that the somatostatin receptors SSTR2 and SSTR3 are present on the parasite egg and worms. We therefore hypothesize that somatostatin reduces either the number of parasite eggs or the secretion of fibrosis inducing-mediators. Our data suggest somatostatin may have therapeutic potential in S. mansoni mediated liver pathology

Can somatostatin control acute bleeding from oesophageal varices in Schistosoma mansoni patients?[ISRCTN63456799]

BACKGROUND: Management of patients with bleeding oesophageal varices comprises of mainly diagnostic endoscopy, sclerotherapy and band ligation. One of the major problems to do any of the above is the active bleeding which makes any intervention difficult. The neuropeptide hormone somatostatin administered exogenously has caused a reduction in portal hypertension and variceal bleeding in patients suffering from liver cirrhosis. We believe that the symptomatic use of somatostatin for variceal bleeding in Schistosoma mansoni infected subjects can reduce bleeding, thereby alleviating the pathology caused by schistosomiasis. METHODS/DESIGN: We herein present a study protocol for establishing this neuropeptide as a potential therapeutic agent in schistosomiasis. Adolescent subjects, age range varying from 12–17 years will be selected, based on several inclusion criteria, most important being infection with Schistosoma mansoni with bleeding from oesophageal varices in the last 24 hours. One group of schistosomiasis patients will be treated with somatostatin and praziquantel, the other with propanolol and praziquantel. Survival graphs will be set up to correlate somatostatin administration with survival time. A two part questionnaire will be set up to control treatment outcomes. The pre-treatment part of the clinical questionnaire will identify inclusion criteria questions, the post-treatment part of the questionnaire will identify treatment outcomes. DISCUSSION: We expect that the administration of somatostatin as a bolus followed by a 24 hour long infusion, will stop bleeding immediately, delay rebleeding as compared to the control study group and delay mortality in the somatostatin treated subjects

Oxidative modification of low-density lipoproteins and the outcome of renal allografts at 11/2 years

Oxidative modification of low-density lipoproteins and the outcome of renal allografts at 11/2 years.BackgroundPrevious studies reported a significant association between hyperlipidemia of the recipient and chronic allograft nephropathy (CAN). However, the nature and the pathogenic mechanism of circulating lipid abnormalities in CAN remain unclear.MethodsIn a prospective study of 50 consecutive adult recipients of a cadaveric renal allograft, we investigated the impact of lipid abnormalities on the outcome of the graft at 11/2 years. Besides morphometric analysis of implantation and protocol biopsies, clinical and biochemical variables were studied at three-month intervals. Plasma concentrations of oxidized low-density lipoprotein (OxLDL) were determined by means of enzyme-linked immunosorbent assay. Immunohistochemical staining for OxLDL and macrophages was performed on paired renal biopsies. Study end points were the fractional interstitial volume and the 24-hour creatinine clearance at 11/2 years.ResultsHigh-density lipoprotein (HDL) cholesterol of the recipient ≤47 mg/dL was a risk factor for the functional (RR = 1.56; 95% CI, 0.978 to 2.497) and the morphological (RR = 2.75; 95% CI, 1.075 to 7.037) outcome of the graft, mainly in patients without acute rejection (RR = 2.03; 95% CI, 1.13 to 3.65, and RR = 4.67; 95% CI, 1.172 to 18.582, respectively). Interstitial accumulation of OxLDL was inversely associated with HDL cholesterol (R = -0.476, P = 0.019), and was associated with a higher density of tubulointerstitial macrophages (R = 0.656, P = 0.001) and a higher fractional interstitial volume at 11/2 years (P = 0.049).ConclusionDecreased HDL cholesterol levels of the recipient adversely affect the outcome of renal allografts through the accumulation of OxLDL in the renal interstitium of the graft. Interstitial accumulation of OxLDL was associated with the presence of macrophages and the development of interstitial fibrosis

MR imaging of clear cell sarcoma (malignant melanoma of the soft parts): A multicenter correlative MRI-pathology study of 21 cases and literature review

Objective. To evaluate MR imaging and pathology findings in order to define the characteristic features of clear cell sarcoma of the soft tissues (malignant melanoma of the soft parts). Design and patients. MR examinations of 21 patients with histologically proven clear cell sarcoma of the musculoskeletal system were retrospectively reviewed and assessed for shape, homogeneity, delineation, signal intensities on T1- and T2-weighted images, contrast enhancement, relationship with adjacent fascia or tendon, secondary bone involvement, and intratumoral necrosis. In 19 cases the pathology findings were available for review and for a comparative MR-pathology study. Results. On T1-weighted images, lesions were isointense (n=3), hypointense (n=7) or slightly hyperintense to muscle (n=11). Immunohistochemical examination was performed in 17 patients. All 17 specimens showed positivity for HMB-45 antibody. In nine of 11 lesions with slightly increased signal intensity on T1-weighted images, a correlative MR imaging-pathology study was possible. All nine were positive to HMB-45 antibody. Conclusions. Clear cell sarcoma of the musculoskeletal system often has a benign-looking appearance on MR images. In up to 52% of patients, this lesion with melanocytic differentiation has slightly increased signal intensity on T1-weighted images compared with muscle. As the presence of this relative higher signal intensity on T1-weighted images is rather specific for tumors displaying melanocytic differentiation, radiologists should familiarize themselves with this rare entity and include it in their differential diagnosis when confronted with a well- defined, homogeneous, strongly enhancing mass with slightly higher signal intensity compared with muscle on native T1-weighted images

An Autopsy Study Describing Causes of Death and Comparing Clinico-Pathological Findings among Hospitalized Patients in Kampala, Uganda

Background: Information on causes of death in HIV-infected patients in Sub-Saharan Africa is mainly derived from observational cohort and verbal autopsy studies. Autopsy is the gold standard to ascertain cause of death. We conducted an autopsy study to describe and compare the clinical and autopsy causes of death and contributory findings in hospitalized HIV-infected and HIV-uninfected patients in Uganda. Methods: Between May and September 2009 a complete autopsy was performed on patients that died on a combined infectious diseases gastroenterology ward in Mulago Hospital in Kampala, Uganda. Autopsy cause of death and contributing findings were based on the macro- and microscopic post-mortem findings combined with clinical information. Clinical diagnoses were reported by the ward doctor and classified as confirmed, highly suspected, considered or not considered, based on information derived from the medical chart. Results are reported according to HIV serostatus. Results: Fifty-three complete autopsies were performed in 66 % HIV-positive, 21 % HIV-negative and 13 % patients with an unknown HIV serological status. Infectious diseases caused death in 83 % of HIV-positive patients, with disseminated TB as the main diagnosis causing 37 % of deaths. The spectrum of illness and causes of death were substantially different betwee

Identification of cell-of-origin breast tumor subtypes in inflammatory breast cancer by gene expression profiling

Inflammatory breast cancer (IBC) is an aggressive form of locally advanced breast cancer with high metastatic potential. Most patients have lymph node involvement at the time of diagnosis and 1/3 of the patients have distant metastases. In a previous study, we demonstrated that IBC is a distinct form of breast cancer in comparison with non-IBC. The aim of this study was to investigate the presence of the different molecular subtypes in our data set of 16 IBC and 18 non-IBC specimen. Therefore, we selected an ‘intrinsic gene set’ of 144 genes, present on our cDNA chips and common to the ‘intrinsic gene set’ described by Sorlie et al. [PNAS, 2003]. This set of genes was tested for performance in the Norway/Stanford data set by unsupervised hierarchical clustering. Expression centroids were then calculated for the core members of each of the five subclasses in the Norway/Stanford data set and used to classify our own specimens by calculating Spearman correlations between each sample and each centroid. We identified the same cell-of-origin subtypes in IBC as those already described in non-IBC. The classification was in good agreement with immunohistochemical data for estrogen receptor protein expression and cytokeratin 5/6 protein expression. Confirmation was done by an alternative unsupervised hierarchical clustering method. The robustness of this classification was assessed by an unsupervised hierarchical clustering with an alternative gene set of 141 genes related to the cell-of-origin subtypes, selected using a discriminating score and iterative random permutation testing. The contribution of the different cell-of-origin subtypes to the IBC phenotype was investigated by principal component analysis. Generally, the combined ErbB2-overexpressing and basal-like cluster was more expressed in IBC compared to non-IBC, whereas the combined luminal A, luminal B and normal-like cluster was more pronounced in non-IBC compared to IBC. The presence of the same molecular cell-of-origin subtypes in IBC as in non-IBC does not exclude the specific molecular nature of IBC, since gene lists that characterize IBC and non-IBC are entirely different from gene lists that define the different cell-of-origin subtypes, as evidenced by principal component analysis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44236/1/10549_2005_Article_9015.pd

The Isolation of Nucleic Acids from Fixed, Paraffin-Embedded Tissues–Which Methods Are Useful When?

Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase “blocks” during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims