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

Increasing frailty is associated with higher prevalence and reduced recognition of delirium in older hospitalised inpatients: results of a multi-centre study

Purpose: Delirium is a neuropsychiatric disorder delineated by an acute change in cognition, attention, and consciousness. It is common, particularly in older adults, but poorly recognised. Frailty is the accumulation of deficits conferring an increased risk of adverse outcomes. We set out to determine how severity of frailty, as measured using the CFS, affected delirium rates, and recognition in hospitalised older people in the United Kingdom. Methods: Adults over 65 years were included in an observational multi-centre audit across UK hospitals, two prospective rounds, and one retrospective note review. Clinical Frailty Scale (CFS), delirium status, and 30-day outcomes were recorded. Results: The overall prevalence of delirium was 16.3% (483). Patients with delirium were more frail than patients without delirium (median CFS 6 vs 4). The risk of delirium was greater with increasing frailty [OR 2.9 (1.8–4.6) in CFS 4 vs 1–3; OR 12.4 (6.2–24.5) in CFS 8 vs 1–3]. Higher CFS was associated with reduced recognition of delirium (OR of 0.7 (0.3–1.9) in CFS 4 compared to 0.2 (0.1–0.7) in CFS 8). These risks were both independent of age and dementia. Conclusion: We have demonstrated an incremental increase in risk of delirium with increasing frailty. This has important clinical implications, suggesting that frailty may provide a more nuanced measure of vulnerability to delirium and poor outcomes. However, the most frail patients are least likely to have their delirium diagnosed and there is a significant lack of research into the underlying pathophysiology of both of these common geriatric syndromes

Some Surprising Results About Covariate Adjustment in Logistic Regression Models

Results from classic linear regression regarding the effect of adjusting for covariates upon the precision of an estimator of exposure effect are often assumed to apply more generally to other types of regression models. In this paper we show that such an assumption is not justified in the case of logistic regression, where the effect of adjusting for covariates upon precision is quite different. For example, in classic linear regression the adjustment for a non-confounding predictive covariate results in improved precision, whereas such adjustment in logistic regression results in a loss of precision. However, when testing for a treatment effect in randomized studies, it is always more efficient to adjust for predictive covariates when logistic models are used, and thus in this regard the behavior of logistic regression is the same as that of classic linear regression

Segmental Dilation of the Colon: Support for a Muscle Wall Etiology

A patient with segmental dilatation of the colon who presented with chronic constipation is reported. A review of previous cases of this rarely reported condition and the typical features of the disorder is presented. A possible contributing factor to its etiology is proposed on the basis of a pathological appearance of the colonic muscle wall not previously described

Hepatic Giant Cell Arteritis and Polymyalgia Rheumatica

Polymyalgia rheumatica (PMR) is a clinical syndrome of the elderly characterized by malaise, proximal muscle aching and stiffness, low grade fever, elevated erythrocyte sedimentation rare and the frequent association with temporal giant cell arteritis. The authors describe a case of PMR associated with hepatic giant cell arteritis. This lesion has been described in two other clinical reports. The distribution of the arteritis may be patchy; in this report, diagnosis was made with a wedge biopsy performed after an initial nonspecific percutaneous liver biopsy. The authors review the spectrum of liver involvement in PMR and giant cell arteritis. Hepatic abnormalities respond to systemic corticosteroids, and patients with hepatic arteritis have a good prognosis

Graft-Versus-Host Disease after Liver Transplantation Complicated by Systemic Aspergillosis with Pancarditis

Acute graft-versus-host disease (GVHD) is a common complication after bone marrow transplantation, with characteristic rash and diarrhea being the most common features. After liver transplantation, however, this phenomenon is very rare. Most transplant patients are on a variety of medications, including immunosuppressants; therefore, the differential diagnosis of skin rash or diarrhea is broad. A 37-year-old man who underwent liver transplantation for primary biliary cirrhosis, and developed a rash and watery diarrhea, is presented. Skin and colonic biopsies confirmed acute GVHD. A pulse of intravenous steroids was given. The skin rash improved, but he developed pancytopenia. His course was complicated by central line infection, jugular and subclavian vein thrombosis, pseudomembranous colitis, recurrent bacteremia, cholestasis on total parenteral nutrition and cytomegalovirus infection. After the onset of pleuritic chest pain and clinical sepsis, spiral computed tomography scan of his chest and abdomen revealed septic infarcts in multiple organs. Despite empirical treatment with amphotericin B, he died of multiorgan dysfunction syndrome within 72 h. Autopsy revealed systemic aspergillosis with pancarditis, endocardial vegetations, and septic pulmonary, splenic, hepatic and renal infarcts. The pathogenesis and experience with this rare, but often fatal, complication of liver transplantation are reviewed. In contrast to GVHD after bone marrow transplantation, pancytopenia is common and liver dysfunction is rare. One should have a high level of suspicion in the liver transplant recipient presenting with rash and/or diarrhea

Familial enteropathy with villous edema and immunoglobulin G2 subclass deficiency

We describe a familial form of recurrent acute, life-threatening secretory diarrhea associated with distinctive jejunal histologic changes and IgG2 subclass deficiency. Symptoms begin abruptly with anorexia and vomiting, and progress within hours to massive secretory diarrhea and shock with profound neutropenia and hypoproteinemia, including hypoalbuminemia and hypogammaglobulinemia. Affected survivors recover quickly and thereafter grow and develop normally. Biopsy specimens obtained during remission from 3 adults and 11 children show club-shaped jejunal villi broadened by edema and histiocytes with imbibed fluid; the overlying intestinal epithelium and brush border appear normal, but the basement membrane is interrupted in some areas. No characteristic microorganisms have been identified in association with the syndrome. Clinical manifestations cease in the second decade, but the abnormal jejunal histologic pattern persists into adult life. Female and male patients are equally affected, although all fatal cases have been in female subjects. Inheritance appears dominant with variable penetrance: one family member without a history of diarrhea has characteristic biopsy findings and another appears to be an obligate carrier with normal biopsy findings. Affected individuals have a reduced serum concentration of IgG2. We believe that this familial enteropathy is a unique entity, not previously described. (J P EDIATR 1994;125:541-8

Graft-versus-host disease after liver transplantation complicated by systemic aspergillosis with pancarditis BRIEF COMMUNICATION Romagnuolo et al

. Graftversus-host disease after liver transplantation complicated by systemic aspergillosis with pancarditis. Can J Gastroenterol 2000;14(7):637-640. Acute graft-versus-host disease (GVHD) is a common complication after bone marrow transplantation, with characteristic rash and diarrhea being the most common features. After liver transplantation, however, this phenomenon is very rare. Most transplant patients are on a variety of medications, including immunosuppressants; therefore, the differential diagnosis of skin rash or diarrhea is broad. A 37-year-old man who underwent liver transplantation for primary biliary cirrhosis, and developed a rash and watery diarrhea, is presented. Skin and colonic biopsies confirmed acute GVHD. A pulse of intravenous steroids was given. The skin rash improved, but he developed pancytopenia. His course was complicated by central line infection, jugular and subclavian vein thrombosis, pseudomembranous colitis, recurrent bacteremia, cholestasis on total parenteral nutrition and cytomegalovirus infection. After the onset of pleuritic chest pain and clinical sepsis, spiral computed tomography scan of his chest and abdomen revealed septic infarcts in multiple organs. Despite empirical treatment with amphotericin B, he died of multiorgan dysfunction syndrome within 72 h. Autopsy revealed systemic aspergillosis with pancarditis, endocardial vegetations, and septic pulmonary, splenic, hepatic and renal infarcts. The pathogenesis and experience with this rare, but often fatal, complication of liver transplantation are reviewed. In contrast to GVHD after bone marrow transplantation, pancytopenia is common and liver dysfunction is rare. One should have a high level of suspicion in the liver transplant recipient presenting with rash and/or diarrhea. Réaction du greffon contre l'hôte après transplantation hépatique compliquée par une aspergillose systémique et une pancardite RÉSUMÉ : La réaction aiguë du greffon contre l'hôte est une complication courante après la transplantation de moelle osseuse, qui se manifeste typiquement par des éruptions cutanées et de la diarrhée. Après la transplantation hépatique, par contre, ce phénomène est très rare. La plupart des receveurs de transplantation prennent divers médicaments dont des immunosuppresseurs. Le diagnostic différentiel des éruptions cutanées ou de la diarrhée est donc vaste. On présente ici le cas d'un homme de 37 ans qui a subi une transplantation hépatique pour cirrhose biliaire primaire et qui a développé une éruption cutanée et de la diarrhée. Les biopsies cutanées et coloniques ont confirmé une réaction aiguë du greffon contre l'hôte. Des corticostéroïdes par voie intraveineuse ont été administrés en bolus. L'éruption cutanée s'est améliorée, mais le patient a développé une pancytopénie. Son état s'est compliqué d'une infection de son cathéter central, d'une thrombose veineuse jugulaire et sous-clavière, d'une colite voir page suivante G raft-versus-host disease (GVHD) is a common complication of bone marrow transplantation (BMT) and involves donor lymphocytes attacking the host and leading to a constellation of symptoms and signs, including diarrhea, skin rash and elevated liver function tests. After orthotopic liver transplantation (OLT), however, this phenomenon is quite rare, has a slightly different presentation and is associated with a high sepsis-related mortality. Immunosuppression of the host is an important prerequisite for GVHD; however, further immunosuppression is the standard approach, in hopes of suppressing or destroying the donor lymphocytes out of proportion to those of the host. The following case outlines the presentation, treatment and approach of complications in a liver transplant recipient with GVHD. CASE PRESENTATION A 37-year-old married man with primary biliary cirrhosis, hypercholesterolemia and type II diabetes mellitus presented with a one-week history of skin rash and diarrhea 23 days after having undergone OLT. The rash was a pruritic, symmetrical, erythematous, maculopapular skin eruption on his neck, back and chest, with prominent involvement of his palms and soles. The rash was perifollicular, and there were some coalescing patches of erythema and target lesions. As well, he complained of three days of large-volume, watery, nonbloody diarrhea with urgency and tenesmus, but denied having fever, abdominal pain or nausea. His other medications included ranitidine, insulin, prednisone 20 mg orally once daily, and cyclosporine 375 mg orally bid, with an admission trough level of 470 m g/L. In addition, he had been enrolled in a double-blinded study receiving either placebo or basiliximab, a chimeric (mouse/human) anti-interleukin (IL)-2 receptor monoclonal antibody. The OLT surgery went without complication A skin punch biopsy was consistent with GVHD. His creatinine level was 410 m mol/L and fell to 144 m mol/L with intravenous fluids. Sigmoidoscopy and gastroscopy revealed 30 cm of severe colitis with multiple shallow ulcers, ulcerative esophagitis, gastritis and duodenitis. All biopsies revealed evidence of acute GVHD, with apoptosis and glandular dropout After one week, he developed thrombocytopenia (62x10 9 /L), and intravenous methylprednisolone sodium succinate 500 mg was given daily and then slowly tapered. After an additional week, his rash improved but the diarrhea persisted. A dose of intravenous immunoglobulin was given. His course was complicated by pseudomembranous colitis, recurrent Gram-negative bacteremia, jugular/subclavian vein thrombosis related to line infection, CMV disease, severe pancytopenia (requiring platelets and granulocyte colony-stimulating factor) and cholestasis (total parenteral nutrition-related). Forty-two days after admission, as he began to improve, he started having brief episodes of pleuritic right-sided chest pain without shortness of breath. A chest x-ray revealed an infiltrate below the right hilum and small nodular opacities peripherally in the left midlung zone. Lung biopsy could not be performed because of refractory thrombocytopenia. He quickly deteriorated and blood cultures grew Enterobacter species. With the suspicion of systemic aspergillosis, intravenous amphotericin B was begun and his cyclosporine was held. After an asystolic cardiac arrest, he was resuscitated and a pacemaker was inserted. He underwent an unrevealing bronchoscopy (no fungi, no pneumocystis), but a spiral computed tomography was consistent with multiple pulmonary embol