Showing papers in "Journal of Clinical Pathology in 2001"

Antioxidants in health and disease

Abstract: Free radical production occurs continuously in all cells as part of normal cellular function. However, excess free radical production originating from endogenous or exogenous sources might play a role in many diseases. Antioxidants prevent free radical induced tissue damage by preventing the formation of radicals, scavenging them, or by promoting their decomposition. This article reviews the basic chemistry of free radical formation in the body, the consequences of free radical induced tissue damage, and the function of antioxidant defence systems, with particular reference to the development of atherosclerosis.

Method for the measurement of antioxidant activity in human fluids

Abstract: Aim —To develop a new, simple, and cheap method for estimating antioxidant activity in human fluids. Methods —The assay measured the capacity of the biological fluids to inhibit the production of thiobarbituric acid reactive substances (TBARS) from sodium benzoate under the influence of the free oxygen radicals derived from Fenton's reaction. A solution of 1 mmol/litre uric acid was used as standard. Results —The following mean (SD) antioxidative activities were found (as uric acid) in the various biological fluids: serum, 2.04 (0.20) mmol/litre; urine, 176.5 (25.6) μmol/litre; cerebrospinal fluid, 95.0 (26.9) μmol/litre; aqueous humour oculi, 61.25 (9.9) μmol/litre; saliva, 838.5 (48.2) μmol/litre; tears, 247.0 (17.0) μmol/litre; ascites fluid, 270.0 (63.3) μmol/litre; kidney cyst fluid, 387.1 (28.1) μmol/litre. Small samples of the biological material were needed for the analyses: 10 μl of serum and 50–100 μl of other body fluids. In the sera of 48 healthy individuals there was a significant positive correlation between values obtained with the Randox method (as a reference method) and the new method proposed here (correlation coefficient, 0.8728; mean difference between methods, <0.4%). Conclusions —This method is easy, rapid, reliable, and practical for the routine measurement of total antioxidant activity in serum and other human body fluids. Small samples of biological material are needed for the analyses and the results are comparable with the reference (Randox) method.

New insights into the role of cytokines in asthma

Abstract: Asthma is a triad of intermittent airway obstruction, bronchial smooth muscle cell hyperreactivity to bronchoconstrictors, and chronic bronchial inflammation. From an aetiological standpoint, asthma is a heterogeneous disease, but often appears as a form of immediate hypersensitivity. Many patients with asthma have other manifestations of atopy, such as rhinitis or eczema. Even among non-atopic patients with asthma, the pathophysiology of airway constriction is similar, raising the hypothesis that alternative mechanisms of mast cell degranulation may underlie the disease. The primary inflammatory lesion of asthma consists of accumulation of CD4(+) T helper type 2 (TH2) lymphocytes and eosinophils in the airway mucosa. TH2 cells orchestrate the asthmatic inflammation through the secretion of a series of cytokines, particularly interleukin 4 (IL-4), IL-13, IL-5, and IL-9. IL-4 is the major factor regulating IgE production by B cells, and is required for optimal TH2 differentiation. However, blocking IL-4 is not sufficient to inhibit the development of asthma in experimental models. In contrast, inhibition of IL-13, another TH2 cytokine whose signal transduction pathway overlaps with that of IL-4, completely blocks airway hyperreactivity in mouse asthma models. IL-5 is a key factor for eosinophilia and could therefore be responsible for some of the tissue damage seen in chronic asthma. IL-9 has pleiotropic activities on allergic mediators such as mast cells, eosinophils, B cells and epithelial cells, and might be a good target for therapeutic interventions. Finally, chemokines, which can be produced by many cell types from inflamed lungs, play a major role in recruiting the mediators of asthmatic inflammation. Genetic studies have demonstrated that multiple genes are involved in asthma. Several genome wide screens point to chromosome 5q31--33 as a major susceptibility locus for asthma and high IgE values. This region includes a cluster of cytokine genes, and genes encoding IL-3, IL-4, IL-5, IL-9, IL-13, granulocyte macrophage colony stimulating factor, and the beta chain of IL-12. Interestingly, for some of these cytokines, a linkage was also established between asthma and their receptor. Another susceptibility locus has been mapped on chromosome 12 in a region that contains other potential candidate cytokine genes, including the gene encoding interferon gamma, the prototypical TH1 cytokine with inhibitory activities for TH2 lymphocytes. Taken together, both experimental and genetic studies point to TH2 cytokines, such as IL-4, IL-13, IL-5, and IL-9, as important targets for therapeutic applications in patients with asthma.

Overwhelming infection in asplenic patients: current best practice preventive measures are not being followed.

Abstract: Aims—Patients without spleens are at increased risk of overwhelming infection. Recently, greater efforts, including the publication of national guidelines, have been made to improve the management of asplenic individuals. In theory, risks of serious sepsis can be reduced by good advice, immunisation, and antibiotic prophylaxis. In practice, such preventive measures might not be followed or may fail. A study of recent cases of overwhelming postsplenectomy infection (OPSI) was undertaken to examine specific associated factors and to determine whether currently recommended preventive measures are being followed. Methods—Cases of OPSI were identified and reported mainly by microbiologists across the country using a specifically designed proforma. Data including the nature of the infection and vaccination/antibiotic prophylaxis history since splenectomy were obtained. Results—Seventy seven cases were reported. The age range varied from 3 months (congenital asplenia) to 87 years. In those who had undergone surgical splenectomy, the time interval between surgery and OPSI varied from 24 days to 65 years. Overall mortality reached 50%, with underlying haematological malignancy associated with the highest death rate. Streptococcus pneumoniae caused approximately 90% episodes. Only 31% individuals had received pneumococcal vaccination before OPSI. Seven of 17 pneumococcal infections in immunised cases could be considered vaccine failures. Few patients had been adequately advised on antibiotic prophylaxis or other measures. Conclusions—Currently accepted best practice for managing asplenic patients is not being followed. Some OPSI cases may still be preventable but many asplenic individuals remain unrecognised. The compilation of asplenic patient registers might help to implement agreed policies with audit necessary to evaluate compliance. More is needed to ensure optimal management for this cohort of the population. Key Words: splenectomy • immunisation • overwhelming postsplenectomy infection • asplenic patient register

The histopathological differential diagnosis of gastrointestinal stromal tumours.

Abstract: Gastrointestinal stromal tumours (GISTs), initially presumed to be of "true" smooth muscle origin, encompass a heterogeneous, and as yet incompletely understood, group of mesenchymal tumours with respect to their origin, cellular differentiation, and prognosis. Cellular morphology ranges from predominantly spindle shaped to epithelioid in character, whereas differentiation pathways, as determined primarily by immunohistochemistry and ultrastructure, can vary from indeterminate to myoid and/or neural. Recent work has indicated that the interstitial cells of Cajal, a complex cellular network postulated to act as pacemaker cells of the gastrointestinal tract, which exhibit both myoid and neural features, could be candidates for tumour histogenesis. This would provide a plausible and attractive explanation for the variable differentiation pathways identified in the GIST category to date. Nevertheless, the occasional but undisputed location of GISTs outside the gastrointestinal tract (omentum, peritoneum, and retroperitoneum) might mitigate against such an origin, and their histogenesis remains open to debate. The c-kit proto-oncogene, encoding a growth factor receptor with tyrosine kinase activity, has been postulated to play an important role in tumorigenesis because "gain of function" mutations in this gene, localised to chromosome 4q11-21, are being increasingly identified in hereditary and sporadic cases. Monoclonal and polyclonal antibodies directed at the c-kit gene product expressed on the cell surface (CD117/c-kit) appear to be increasingly helpful in resolving the histopathological differential diagnosis between GISTs and true gastrointestinal smooth muscle neoplasms, schwannomas, and other far less frequently occurring mesenchymal tumours at this site. Although tumours with a clinically benign course appear to be more common than their malignant counterparts, no specific histological criteria have as yet been identified to enable an unambiguous prediction of biological behaviour. Increasing tumour size and mitotic activity favour aggressive tumour behaviour, whereas the prognostic value of germline and somatic mutations within the c-kit proto-oncogene remains to be elucidated further. It is the aim of this synopsis to highlight the relevant fundamental and diagnostic developments with respect to this complex group of neoplasms.

Evidence of type II pneumocyte apoptosis in the pathogenesis of idiopathic pulmonary fibrosis (IFP)/usual interstitial pneumonia (UIP)

Abstract: Background/Aims—The pathogenesis of idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP), a chronic and incurable human respiratory disease, is not well established. This study was designed to investigate whether the apoptosis of type II pneumocytes could be the precipitating factor in the pathogenesis of IPF. Methods—Nineteen specimens obtained by retrospective review of the medical and pathological records of 55 patients with IPF, four normal subjects, and 10 disease control lungs were analysed. The selected specimens had normal alveoli with intervening patchy scarring of the lung parenchyma, fulfilling the pathological criteria for UIP. To identify individual cells undergoing apoptosis in the normal alveoli, electron microscopy and in situ end labelling of fragmented DNA were performed on paraffin wax embedded sections using digoxigenin-11-dUTP and the enzyme terminal deoxynucleotidyl transferase. Results—Apoptosis was detected in the normal alveoli of 17 of the 19 patients with IPF/UIP and was absent in the controls. Electron microscopy demonstrated apoptotic changes in type II pneumocytes. These results indicate that apoptotic type II pneumocyte death occurs in normal alveoli of IPF/UIP and could be the principal cause of several events that account for the histological, clinical, and functional alterations seen in IPF/UIP. Conclusions—In conclusion, numerous type II pneumocytes from the normal alveoli of most patients with IPF/UIP actively undergo programmed cell death. This finding may shed new light on the pathogenesis of this disease, with implications mainly for the treatment of affected patients. Key Words: apoptosis • type II pneumocytes • idiopathic pulmonary fibrosis • pathogenesis

Pulmonary preinvasive neoplasia

Abstract: Advances in molecular biology have increased our knowledge of the biology of preneoplastic lesions in the human lung. The recently published WHO lung tumour classification defines three separate lesions that are regarded as preinvasive neoplasia. These are (1) squamous dysplasia and carcinoma in situ (SD/CIS), (2) atypical adenomatous hyperplasia (AAH), and (3) diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIP-NECH). SD/CIS is graded in four stages (mild, moderate, severe, and CIS), based upon the distribution of atypical cells and mitotic figures. Most airways showing SD/CIS demonstrate a range of grades; many epithelia are hard to assess and the reproducibility of this complex system remains to be established. Detailed criteria are, however, welcome and provide an objective framework on which to compare various molecular changes. Alterations in gene expression and chromosome structure known to be associated with malignant transformation can be demonstrated in CIS, less so in dysplasias, but also in morphologically normal epithelium. The changes might be sequential, and their frequency and number increase with atypia. Less is known of the "risk of progression" of SD/CIS to invasive "central" bronchial carcinoma. It may take between one and 10 years for invasion to occur, yet the lesion(s) may be reversible if carcinogen exposure ceases. AAH may be an important precursor lesion for peripheral "parenchymal" adenocarcinoma of the lung: the "adenoma" in an adenoma-carcinoma sequence. There is good morphological evidence that AAH may progress from low to high grade to bronchioloalveolar carcinoma (BAC; a non-invasive lesion by definition). Invasion then develops within BAC and peripheral lung adenocarcinoma evolves. The molecular events associated with this progression are not well understood and studies are hampered by a lack of clear criteria to distinguish high grade AAH from BAC. Nonetheless, as with SD/CIS, the patterns of expression of tumour associated genes are consistent with neoplastic progression. We have little idea of the incidence of AAH in the normal or "smoking" populations. It is found more frequently in cancer bearing lungs, especially in those with adenocarcinoma, and is more common in women. No data are available on the risk of progression of AAH. DIPNECH is an exceptionally rare lesion associated with the development of multiple carcinoid tumours. Almost nothing is known of its biology. Knowledge of these lesions will be crucial in the design and understanding of lung cancer screening programmes, where it is likely that the morphological and, more importantly perhaps, the molecular characteristics of these lesions will provide useful targets for detection and possibly even treatment.

Brush cytology in the assessment of pancreatico-biliary strictures: a review of 406 cases.

Abstract: Aims—To assess the accuracy of brush cytology in patients investigated for pancreatico‐biliary strictures. Methods—All pancreatico‐biliary brush cytology specimens submitted from two major teaching hospitals over a 6.5 year period were reviewed. Four hundred and forty eight satisfactory specimens from 406 patients with adequate clinical and/or pathological follow up data were examined in the study period. Results—Two hundred and forty six patients (60.6%) were shown to have neoplastic strictures. One hundred and forty seven tumours were identified cytologically, including 87 of 146 pancreatic carcinomas, 29 of 47 cholangiocarcinomas, one of one bile duct adenoma, four of seven carcinomas of the gallbladder, eight of 13 ampullary carcinomas, two of three ampullary adenomas, 10 of 16 malignancies of undetermined origin, none of two islet cell tumours, one of three hepatocellular carcinomas, and five of eight metastatic tumours. The three adenomas identified on brush cytology could not be distinguished from adenocarcinoma morphologically. One hundred and sixty patients (39.4%) had benign strictures, most often as a result of chronic pancreatitis and bile duct stones. There were three false positive cytological diagnoses mainly as a result of the misinterpretation of cases with relatively scant and/or degenerative atypical epithelial cells. Forty one cases were reported as atypical or suspicious of malignancy on brush cytology, of which 29 were ultimately shown to have carcinoma. The overall diagnostic sensitivity and specificity were 59.8% and 98.1%, respectively. The sensitivity increased from 44.3% in the initial third of cases to 70.7% in the final third of cases examined in the series. Conclusions—Brush cytology, in conjunction with other clinical and radiological investigations, is a useful technique in the assessment of patients with suspected pancreatico‐biliary neoplasia. (J Clin Pathol 2001;54:449‐455)

The prognostic relevance of angiogenesis and mast cells in squamous cell carcinoma of the oesophagus

Abstract: Aims —Angiogenesis, an important prognostic factor in several tumours, is a complex event mediated by angiogenic factors released from cancer cells and host immune cells. Among the host immune cells, a role has been implicated for mast cells in tumour progression via promoting angiogenesis. Data have been recorded that indicate a correlation between intratumoral neovascularisation, as assessed by microvessel density (MVD), and prognosis in squamous cell carcinoma (SCC) of the oesophagus. However, a correlation between mast cell density (MCD) and either prognosis or angiogenesis has not been delineated yet in this disease. The aim of this study was to investigate the prognostic value of MVD and MCD in SCC of the oesophagus. The correlation between MVD and MCD was also evaluated. Methods —MVD and MCD were investigated in tumour specimens from 53 patients diagnosed with SCC of the oesophagus. Intratumoral microvessels were stained with anti-CD34 antibody and mast cells with toluidine blue before being measured by light microscopy. Results —Both MVD and MCD were associated with the depth of wall invasion, lymph node metastasis, and tumour progression (stage). A significant correlation was noted between MVD and MCD values ( r = 0.72). The prognosis was significantly worse in patients with high MVD (≥ 92) and high MCD (≥ 18) values. Multivariate analysis indicated that MVD and stage were independent predictors of survival. Conclusions —These findings support the suggestion that MVD is a reliable prognostic marker in SCC of the oesophagus. Moreover, MCD may have a role in the angiogenesis of these tumours and might be responsible for their aggressive behaviour.

Bone marrow trephine biopsy.

Abstract: Trephine biopsies of the bone marrow should be carried out, when clinically indicated, by trained individuals following a standard operating procedure. A bone marrow aspiration should be performed as part of the same procedure. For patient safety and convenience, biopsies are usually performed on the posterior iliac crest. The biopsy specimen should measure at least 1.6 cm and, if it does not, consideration should be given to repeating the procedure, possibly on the contralateral iliac crest. If bone marrow aspiration is found to be impossible, imprints from the biopsy specimen should be obtained. Otherwise, the specimen is placed immediately into fixative and after fixation is embedded in a resin or, more usually, decalcified and embedded in paraffin wax. Thin sections are cut and are stained, as a minimum, with haematoxylin and eosin and with a reticulin stain. A Giemsa stain is also desirable. A Perls' stain does not often give useful information and is not essential in every patient. The need for other histochemical or immunohistochemical stains is determined by the clinical circumstances and the preliminary findings. Trephine biopsy sections should be examined and reported in a systematic manner, assessment being made of the bones, the vessels and stroma, and the haemopoietic and any lymphoid or other tissue. Assessment should begin with a very low power objective, the entire section being examined. Further examination is then done with an intermediate and high power objective. Ideally, reporting of trephine biopsy sections should be done by an individual who is competent in both histopathology and haematology, and who is able to make an appropriate assessment of both the bone marrow aspirate and the trephine biopsy sections. When this is not possible, there should be close consultation between a haematologist and a histopathologist. The report should both describe the histological findings and give an interpretation of their importance. A signed or computer authorised report should be issued in a timely manner. If the report is a preliminary, this must be clearly stated.

Vasculitides associated with HIV infection

Abstract: The manifestations of human immunodeficiency virus (HIV) infection are protean and vasculitides are one of the less common but nonetheless important consequences. A wide range of vasculitides can be encountered, ranging from vasculitis resulting from specific infective agents to a non-specific vasculitis. Among the infective causes, cytomegalovirus and tuberculosis are probably the most common. A polyarteritis nodosa-like vasculitis with important differences to classic polyarteritis nodosa is also described. Hypersensitivity vasculitis resulting in several patterns of vasculitis and angiocentric immunoproliferative vasculitis are well recognised. As part of the immunocompromise caused by HIV, a granulomatous inflammation involving small arteries and veins of the brain surface and leptomeninges, termed a primary angiitis of the central nervous system, is a rare vasculitis associated with high mortality. A recently described large vessel (aorta, femorals, carotids) vasculopathy resulting in either multiple aneurysm formation or occlusive disease is seen in young adults. An infective agent is not found but aetiologically some of these lesions might be the result of a leucocytoclastic vasculitis of vasa vasora or periadventitial vessels. A final group of non-specific vasculitides not fitting into any of the characteristic patterns described accounts for the residue of vasculitides associated with HIV.

Bone marrow aspiration.

Abstract: Bone marrow aspiration biopsies are carried out principally to permit cytological assessment but also for immunophenotypic, cytogenetic, molecular genetic, and other specialised investigations. Often, a trephine biopsy is carried out as part of the same procedure. Bone marrow aspirations should be carried out by trained individuals who are aware of the indications, contraindications, and hazards of the procedure. They should follow a standard operating procedure. The operator should have made an adequate assessment of clinical and haematological features to ensure both that appropriate indications exist and that all relevant tests are performed. For the patient's comfort and safety, the posterior iliac crest is generally the preferred site of aspiration. Films of aspirated marrow and, when appropriate, films of crushed particles should be made and labelled. Once thoroughly dry, films should be fixed and stained. As a minimum, a Romanowsky stain and a Perls' stain are required. A cover slip should be applied. The bone marrow films should be assessed and reported in a systematic manner so that nothing of importance is overlooked, using a low power, then intermediate, then high power objective. A differential count should be performed. An interpretation of the findings, in the light of the clinical and haematological features, should be given. The report should be signed or computer authorised, using a secure password, and issued in a timely manner.

Immune responses to tumour antigens: implications for antigen specific immunotherapy of cancer

Abstract: Tumour associated antigens recognised by cellular or humoral effectors of the immune system are potential targets for antigen specific cancer immunotherapy. Different categories of cancer antigens have been identified that induce cytotoxic T lymphocyte (CTL) responses in vitro and in vivo, namely: (1) "cancer testis" (CT) antigens, expressed in different tumours and normal testis, (2) melanocyte differentiation antigens, (3) point mutations of normal genes, (4) self antigens that are overexpressed in malignant tissues, and (5) viral antigens. Clinical studies with peptides and proteins derived from these antigens have been initiated to study the efficacy of inducing specific CTL responses in vivo. Immunological and clinical parameters for the assessment of antigen specific immune responses have been defined—delayed type hypersensitivity (DTH), CTL, autoimmmune, and tumour regression responses. Specific DTH and CTL responses and tumour regression have been observed after the intradermal administration of tumour associated peptides alone. Peptide specific immune reactions were enhanced after using granulocyte macrophage stimulating factor (GM-CSF) as a systemic adjuvant by increasing the frequency of dermal antigen presenting Langerhans cells. Complete tumour regression has been observed in the context of measurable peptide specific CTL. However, in single cases with disease progression after an initial tumour response, either a loss of single antigens targeted by CTL or of the presenting major histocompatibility complex (MHC) class I allele was detected, pointing towards immunisation induced immune escape. Cytokines to modulate antigen and MHC class I expression in vivo are being evaluated to prevent immunoselection. Recently, a new CT antigen, NY-ESO-1, has been identified on the basis of spontaneous antibody responses to tumour associated antigens. NY-ESO-1 appears to be one of the most immunogenic antigens known to date, with spontaneous immune responses observed in 50% of patients with NY-ESO-1 expressing cancers. Clinical studies have been initiated to evaluate the immunogenicity of different NY-ESO-1 constructs to induce both humoral and cellular immune responses in vivo. Key Words: tumour antigens • antigen specific T cell response

Giant cell tumour of tendon sheath (localised nodular tenosynovitis): clinicopathological features of 71 cases.

Abstract: Aims/Background—Giant cell tumour of the tendon sheath (GCTTS) is regarded as the most common neoplasm of the hand that can recur after excision. The objective of this study was to review a series of cases in our department and to determine any clinical or pathological features that might predict the likelihood of recurrence. Methods—Clinical data, obtained from pathology request forms and in patient notes, along with the gross and microscopic appearances of 71 cases of GCTTS were evaluated. Results—Clinical features and pathological features identified were similar to those of previous studies. In comparison with previous studies a higher mitotic count (range, 1–21 mitoses/10 high power fields (HPF); mean, 5/10 HPF) was noted in all cases, irrespective of recurrence and numerous apoptotic bodies (up to 30/10 HPF), mainly formed from osteoclast-like giant cells, were present. Conclusions—GCTTS is a relatively rare soft tissue tumour of uncertain histiogenesis. Mitotic and apoptotic figures are a common feature and do not indicate clinical behaviour. Complete local excision is the treatment of choice. Key Words: giant cell tumour tendon sheath • apoptosis • osteoclasts

Measurement of total antioxidant capacity.

Abstract: The accurate assessment of oxidative stress in biological systems is a problem for all investigators working on the role of free radical damage in disease. Numerous assays have been described to measure various free radical damage products or antioxidant status, and the plethora of available techniques attests to the fact that no ideal method is available. The concept of a single test that might reflect total antioxidant capacity (TAC) is an attractive one, and in this issue Koracevic et al describe one such test.1 Low total antioxidant capacity could be indicative of oxidative stress or increased susceptibility to oxidative damage. The idea of a single measurement of total antioxidant capacity is not a new one. Wayner et al in 1985 described the total radical trapping parameter (TRAP) assay, based on the generation of peroxyl radicals from 2,2`-azobis (2-amidinopropane) dihydrochloride (AAPH).2 After adding AAPH to a …

Ataxia telangiectasia gene mutations in leukaemia and lymphoma

Abstract: Ataxia telangiectasia (AT) is a rare multisystem, autosomal, recessive disease characterised by neuronal degeneration, genome instability, and an increased risk of cancer. Approximately 10% of AT homozygotes develop cancer, mostly of the lymphoid system. Lymphoid malignancies in patients with AT are of both B cell and T cell origin, and include Hodgkin's lymphoma, non-Hodgkin's lymphoma, and several forms of leukaemia. The AT locus was mapped to the chromosomal region 11q22-23 using genetic linkage analysis in the late 1980s and the causative gene was identified by positional cloning several years later. The ATM gene encodes a large protein that belongs to a family of kinases possessing a highly conserved C-terminal kinase domain related to the phosphatidylinositol 3-kinase domain. Members of this kinase family have been shown to function in DNA repair and cell cycle checkpoint control following DNA damage. Recent studies indicate that ATM is activated primarily in response to double strand breaks and may be considered a caretaker of the genome. Most mutations in ATM result in truncation and destabilisation of the protein, but certain missense and splicing errors have been shown to produce a less severe phenotype. AT heterozygotes have a slightly increased risk of breast cancer. Atm deficient mice exhibit many of the symptoms found in patients with AT and have a high frequency of thymic lymphoma. The association between mutation of the ATM gene and a high incidence of lymphoid malignancy in patients with AT, together with the development of lymphoma in Atm deficient mice, supports the proposal that inactivation of the ATM gene may be of importance in the pathogenesis of sporadic lymphoid malignancy. Loss of heterozygosity at 11q22-23 (the location of the ATM gene) is a common event in lymphoid malignancy. Frequent inactivating mutations of the ATM gene have been reported in patients with rare sporadic T cell prolymphocytic leukaemia (T-PLL), B cell chronic lymphocytic leukaemia (B-CLL), and most recently, mantle cell lymphoma (MCL). In contrast to the ATM mutation pattern in AT, the most frequent nucleotide changes in these sporadic lymphoid malignancies were missense mutations. The presence of inactivating mutations, together with the deletion of the normal copy of the ATM gene in some patients with T-PLL, B-CLL, and MCL, establishes somatic inactivation of the ATM gene in the pathogenesis of lymphoid malignancies, and strongly suggests that ATM functions as a tumour suppressor. The presence of missense mutations in the germline of patients with B-CLL has been reported, suggesting that some patients with B-CLL may be constitutional AT heterozygotes. The putative hereditary predisposition of B-CLL, although intriguing, warrants further investigation.

Adrenal lipomatous tumours: a 30 year clinicopathological experience at a single institution

Abstract: Aims—Fatty tumours of the adrenal gland are uncommon and their features have received little attention in the literature. The aim of this study is to analyse the features of adrenal lipomatous tumours. Methods—The histological features of primary adrenal tumours reported over a 30 year period (1970 to 1999) in Queen Mary Hospital, Hong Kong were reviewed and the clinicopathological features of adrenal lipomatous tumours were analysed. Results—Adrenal lipomatous tumours were noted in 20 patients (12 men, eight women), and they accounted for 4.8% of the primary adrenal tumours reported. The adrenal fatty tumours comprised 11 myelolipomas, three lipomas, three teratomas, two angiomyolipomas, and one liposarcoma. Calcification or bone was noted in one third (seven of 20) of the adrenal tumours. In some fatty tumours (myelolipoma and angiomyolipoma), the fatty component may be inconspicuous. This is the first report in the English literature of angiomyolipoma and liposarcoma of the adrenal gland. Conclusions—DiVerent types of fatty tumours were noted in the adrenal gland. A high index of suspicion should be maintained with an aim of surgical treatment for selected patients with large and symptomatic adrenal lipomatous lesions. Histological confirmation is needed for diagnosis. (J Clin Pathol 2001;54:707‐712)

Gastric intestinal metaplasia: subtypes and natural history

Abstract: Background —It has been suggested that the subtyping of intestinal metaplasia in the stomach is useful in stratifying patients with regard to risk of developing gastric cancer. Aim —To determine whether subtyping intestinal metaplasia provided useful information regarding the natural history of intestinal metaplasia. Methods —The study used large cup gastric biopsy specimens from predetermined locations (gastric mapping). Follow up biopsies were obtained at one, two, and/or nine years. Biopsies with intestinal metaplasia were stained with high iron diamine/Alcian blue (HID/AB) to determine whether they expressed neutral mucins, sialomucins, or sulphomucins. Results —Seventy nine patients with intestinal metaplasia were studied and characterised with regard to the most advanced subtype of intestinal metaplasia. The most severe type of intestinal metaplasia was type II in 33 patients and type III in 34 patients. Helicobacter pylori was cured in 67 patients. Follow up showed that changes in type of metaplasia (apparent regression or progression) occurred in both directions and were independent of H pylori status. For example, biopsy sites with “loss” of metaplasia at a follow up visit might have it “reappear” at a subsequent visit. During follow up, no patient developed gastric dysplasia or died from gastric cancer. Conclusion —HID subtyping did not provide useful information to the clinician or the pathologist. The data are consistent with the notion that the pattern, extent, and severity of atrophy with/without intestinal metaplasia is a far more important predictor of increased cancer risk than intestinal metaplasia subtype.

Investigation of CMV disease in immunocompromised patients

Abstract: Cytomegalovirus (CMV) is a recognised cause of morbidity and mortality in immunocompromised individuals. This review will concentrate on recent advances in the understanding of the complex interplay between the host and parasite and the pathological consequences of perturbation of the host immune system. The classic view of CMV as a slowly replicating virus is challenged by recent in vivo findings suggesting that active replication occurs dynamically in the human host, with a doubling time of approximately one day. In addition, CMV load plays a major role in viral pathogenesis, such that increased CMV replication is a significant risk factor for disease in all immunocompromised groups studied to date. These studies focus attention on understanding the virological and immunological determinants of enhanced viral replication and its pathological consequences.

Merkel cell carcinoma can be distinguished from metastatic small cell carcinoma using antibodies to cytokeratin 20 and thyroid transcription factor 1

Abstract: Aim —To investigate whether immunohistochemical staining for cytokeratin 20 (CK20) and thyroid transcription factor 1 (TTF-1) is useful in distinguishing Merkel cell carcinomas (MCCs) from metastatic small cell carcinomas (SCCs). Methods —Eleven cases of MCC and 10 of lung SCC were stained for CK20 and TTF-1. Results —Ten of 11 MCCs stained with the antibody to CK20. None was positive for TTF-1. No SCC stained with anti-CK20 and all stained strongly with anti-TTF-1. Conclusions —The use of both anti-CK20 and anti-TTF-1 can reliably distinguish between MCC and metastatic SCC, thus avoiding the need for a detailed clinical investigation of patients with MCC in whom metastatic SCC must be excluded.

Neuroblastoma tumour genetics: clinical and biological aspects

Abstract: Neuroblastoma tumour cells show complex combinations of acquired genetic aberrations, including ploidy changes, deletions of chromosome arms 1p and 11q, amplification of the MYCN oncogene, and—most frequently—gains of chromosome arm 17q. Despite intensive investigation, the fundamental role of these features in neuroblastoma initiation and progression remains to be understood. Nonetheless, great progress has been made in relating tumour genetic abnormalities to tumour behaviour and to clinical outcome; indeed, neuroblastoma provides a paradigm for the clinical importance of tumour genetic abnormalities. Knowledge of MYCN status is increasingly being used in treatment decisions for individual children, and the clinical value of 1p and 17q data as adjuncts or refinements in risk stratification is under active investigation. Reliable detection of these molecular cytogenetic features should be regarded as mandatory for all new cases at presentation. Key Words: neuroblastoma genetics • 17q gain in neuroblastoma • neuroblastoma: 1p and MYCN • tumour genetics and prognosis

Expression of prolactin receptors in normal, benign, and malignant breast tissue: an immunohistological study

Abstract: Aims —Prolactin plays an important role in the proliferation and differentiation of normal breast epithelium, and possibly in the development of breast carcinoma. The effects of prolactin are mediated by its receptor; thus, alteration in the expression of this receptor could be important in studying the biology of breast cancer. This investigation was aimed at comparing the expression of prolactin receptors in normal, benign, and malignant breast tissue. Material/Methods —The expression of prolactin receptors was studied in paraffin wax embedded sections of 102 breast biopsies (93 female and nine male), using the monoclonal antibody B6.2, and the avidin–biotin immunoperoxidase technique. Six biopsies were normal, 34 had benign lesions, and 62 were malignant. Results —In normal cases, prolactin receptor positivity was seen only on the luminal borders of the epithelial cells lining ducts and acini. In most benign lesions, variable degrees of luminal and cytoplasmic staining were seen. Cells showing apocrine metaplasia and florid regular ductal epithelial hyperplasia were mostly negative. In malignant cases, the staining pattern was mostly cytoplasmic and heterogeneous. Forty one of the 59 carcinomas in women showed a degree of positivity involving 10–100% of the tumour cells. A significant direct correlation was found between prolactin receptor and oestrogen receptor staining when only cases that scored more than 100/300 for the latter receptor, using the H scoring system, were considered (p = 0.0207). No correlation was found between prolactin receptors and progesterone receptors, patient9s age, tumour size, tumour grade, or axillary lymph node status. Conclusions —Prolactin receptors seem to be expressed at different cellular sites in normal, benign, and malignant breast epithelial cells. The receptor is expressed in more than two thirds of female breast carcinomas, suggesting that it may play a role in the pathogenesis of the disease. The positivity is correlated with moderate and strong staining for oestrogen receptors in tissue sections, but not with other prognostic factors.

Neutrophil disorders and their management

Abstract: Neutrophil disorders are an uncommon yet important cause of morbidity and mortality in infants and children. This article is an overview of these conditions, with emphasis on clinical recognition, rational investigation, and treatment. A comprehensive list of references is provided for further reading.

ACP Best Practice No 165: front line tests for the investigation of suspected porphyria.

Abstract: The porphyrias are uncommon disorders of haem biosynthesis and their effective management requires prompt and accurate diagnosis. This article describes methods for the determination of urinary porphobilinogen, urinary and faecal total porphyrins, and total porphyrins in erythrocytes and plasma that are suitable for use in non-specialist laboratories. The selection and interpretation of these methods, and the indications for further, more specialised, investigation are discussed.

Salicylic acid in the serum of subjects not taking aspirin. Comparison of salicylic acid concentrations in the serum of vegetarians, non-vegetarians, and patients taking low dose aspirin.

Abstract: Aims—To determine serum salicylic acid concentrations in non-vegetarians and vegetarians not taking salicylate drugs, and to compare these concentrations with those found in patients taking aspirin, 75 mg daily. Methods—Serum samples were obtained from vegetarians (n = 37) and non-vegetarians (n = 39) not taking salicylate drugs. Non-vegetarians and vegetarians were recruited from the community and from a Buddhist monastery, respectively, in Dumfries and Galloway, Scotland. Patients (n = 14) taking aspirin (75 mg daily) were recruited from the Dumfries diabetic clinic. Serum salicylic acid concentrations were determined using a high performance liquid chromatography method with electrochemical detection. Results—Salicylic acid was detected in every serum sample analysed. Higher serum concentrations of salicylic acid were found in vegetarians than non-vegetarians: median concentrations of 0.11 (range, 0.04–2.47) µmol/litre and 0.07 (range, 0.02–0.20) µmol/litre, respectively; the median of the difference was 0.05 µmol/litre (95% confidence interval for difference, 0.03 to 0.08; p < 0.0001). The median serum concentration of salicylic acid in patients taking aspirin (75 mg daily) was 10.03 (range, 0.23–25.40) µmol/litre, which was significantly higher than that found in non-vegetarians and vegetarians. There was overlap in serum salicylic acid concentrations between the vegetarians and patients taking aspirin. Conclusions—Salicylic acid, a non-steroidal anti-inflammatory drug, is present in fruits and vegetables and is found in higher concentrations in vegetarians than non-vegetarians. This suggests that a diet rich in fruits and vegetables contributes to the presence of salicylic acid in vivo. There is overlap between the serum concentrations of salicylic acid in vegetarians and patients taking aspirin, 75 mg daily. These findings may explain, in part, the health promoting effects of dietary fruits and vegetables. Key Words: salicylic acid • salicylates • diet • low dose aspirin

Detection of progesterone receptor forms A and B by immunohistochemical analysis.

Abstract: Aim—The measurement of progesterone receptors (PR) is recommended as part of the clinical management of breast and endometrial cancers, and immunohistochemistry on formalin fixed tissue is now the method of choice. PR is expressed as two isoforms, PRA and PRB, and although both these proteins are expressed in hormone dependent cancers, there is evidence that a large proportion of tumours express a predominance of one isoform. Therefore, it is essential to document the individual detection of PRA and PRB by the presently available anti-PR antibodies. The aim of this study is to investigate the detection of PR isoforms A and B in formalin fixed, paraffin wax embedded cell lines and tissue sections by immunohistochemistry, using a panel of commercial and in house antibodies to human PR. Methods—PR negative cell lines stably transfected to express only PRA (MCF-7Mll/PRA) or PRB (MDA-MB-231/PRB), and tissue sections of human breast carcinoma and normal endometrium were stained using an immunoperoxidase method. A panel of primary PR specific antibodies was evaluated for ability to detect both PRA and PRB proteins, and for intensity and distribution of positive staining under optimal conditions. Results—Of the 11 antibodies assessed, only four recognised PRA and PRB similarly. Six recognised PRA proteins but were unable to detect PRB expression in the cell lines expressing only PRA or PRB. In tissues expressing high amounts of PRA and PRB, all antibodies tested demonstrated positive PR staining. However, in tissues expressing a predominance of PRB, differential staining patterns were observed, with variations in staining intensity and in the proportion of cells positive for PR. Conclusions—Most PR specific antibodies tested failed to detect PRB in formalin fixed tissue by immunohistochemical techniques, despite their ability to do so by immunoblot analysis. These observations suggest that there are conformational differences between PRA and PRB that mask epitopes on the PRB protein recognised by most anti-PR antibodies. The selection of antibodies that recognise both PRB and PRA in formalin fixed tissue is essential for the accurate evaluation of PR positivity in clinical specimens. Key Words: progesterone receptor isoforms • progesterone receptor antibodies • immunohistochemistry

ACP Best Practice No 167: the laboratory diagnosis of urinary tract infection.

Abstract: Urinary tract infection is common, and it is not surprising that urine specimens make up a large proportion of those samples submitted to the routine diagnostic laboratory. Many of these specimens will show no evidence of infection and several methods can be used to screen out negative samples. Those that grow bacteria need to be carefully assessed to quantify the degree of bacteriuria and hence clinical relevance. To influence treatment, a final report should be produced within 24 hours of specimen receipt, with turnaround times continuously monitored. Much work needs to be done to determine the cost effectiveness involved in processing urine specimens and the evidence base for the final report provided.

Evaluation of extraction methods from paraffin wax embedded tissues for PCR amplification of human and viral DNA.

Abstract: Aim —To evaluate the efficiency of phenol/chloroform, microwave, and Qiagen spin column based DNA extractions from paraffin wax embedded tissue for use in the polymerase chain reaction (PCR). In addition, to assess the reliability of amplifying a housekeeping gene to indicate successful viral DNA extraction. Methods —DNA samples extracted from 20 blocks of cervical carcinoma tissues using the three methods were subjected to PCRs targeting 509 bp and 355 bp of the β globin gene, and 450 bp and 150 bp of human papillomavirus (HPV) DNA. Results —Microwave extraction showed the highest positive rate for β globin PCR, whereas the spin column method was the most efficient for HPV DNA extraction. When the 509 bp β globin and 450 bp HPV PCR results were correlated, two of 10, eight of 12, and nine of 10 β globin positive extractions prepared by means of the phenol/chloroform, microwave, and spin column methods, respectively, yielded HPV DNA of the expected size. For the β globin negative samples, HPV was detected in three of 10, two of eight, and four of 10 samples. Conclusions —HPV DNA extraction was most efficient using the Qiagen spin column and had the highest positive predictive value when a housekeeping gene was used as an indicator of successful viral DNA extraction; the phenol/chloroform method was the least efficient. The potential drawbacks of some extraction methods when using a human housekeeping gene to assess the quality of viral DNA extraction need to be considered.

Quantitative assessment of fibrosis and steatosis in liver biopsies from patients with chronic hepatitis C

Abstract: Backgrounds—Hepatic fibrosis is one of the main consequences of liver disease. Both fibrosis and steatosis may be seen in some patients with chronic hepatitis C and alcoholic liver disease (ALD). Aims—To quantitate fibrosis and steatosis by stereological and morphometric techniques in patients with chronic hepatitis C and compare the results with a control group of patients with ALD.In addition,to correlate the quantitative features of fibrosis with the Ishak modified histological score. Materials and methods—Needle liver biopsies from 86 patients with chronic hepatitis C and from 32 patients with alcoholic liver disease (disease controls) were analysed by stereological and morphometric analyses using the Prodit 5.2 system. Haematoxylin and eosin and Picro-Mallory stained sections were used. The area fractions (AA) of fibrosis, steatosis, parenchyma, and other structures (bile duct and central vein areas) were assessed by stereological method. The mean diameters of fat globules were determined by morphometric analysis. Results—Significant diVerences were found in the AA of fibrosis, including fibrosis within portal tract areas, between chronic hepatitis C patients and those with ALD (mean (SD): 19.14 (10.59) v 15.97 (12.51)). Portal and periportal (zone 1) fibrosis was significantly higher (p = 0.00004) in patients with chronic hepatitis C compared with the control group (mean (SD): 9.04 (6.37) v 3.59 (3.16)). Pericentral fibrosis (zone 3) occurred in both groups but was significantly more pronounced in patients with ALD. These results correlate well with the modified Ishak scoring system. However, in patients with cirrhosis (stage 6) with chronic hepatitis C the AA of fibrosis varied between 20% and 74%. The diameter of fat globules was significantly lower in patients with hepatitis C (p = 0.00002) than the ALD group (mean (SD): 14.44 (3.45) v 18.4 (3.32)). Microglobules were more frequent in patients with chronic hepatitis C than in patients with ALD. In patients with chronic hepatitis C, the fat globules had a zonal distribution in comparison with pan steatosis in ALD. Conclusion—Quantitative, stereological techniques are simple and reliable for evaluating hepatic fibrosis and steatosis in chronic hepatitis C. They are most useful for assessing the origin, location, and the stage of fibrosis. Stereology and morphometry are recommended for the quantitation of fibrosis and steatosis, particularly for the evaluation of new treatment strategies in patients with chronic hepatitis C. (J Clin Pathol 2001;54:461‐465)

High quality RNA isolation from tumours with low cellularity and high extracellular matrix component for cDNA microarrays: application to chondrosarcoma

Abstract: Aims—High quality RNA isolation from cartilaginous tissue is considered diYcult because of relatively low cellularity and the abundance of extracellular matrix rich in glycosaminoglycans and collagens. Given the growing interest and technical possibilities to study RNA expression at a high throughput level, research on tissue with these characteristics is hampered by the lack of an eYcient method for obtaining suYcient amounts of high quality RNA. Methods—This paper presents a robust protocol combining two RNA isolation procedures, based on a combination of Trizol and RNA specific columns, which has been developed to obtain high molecular weight RNA from fresh frozen and stored tissue of normal cartilage and cartilaginous tumours. Using this method, RNA was isolated from normal cartilage, peripheral chondrosarcoma, and central chondrosarcoma. Results—The yields ranged from 0.1 to 0.5 µg RNA/mg tissue. RNA isolated with this method was stable and of high molecular weight. RNA samples from normal cartilage and from two chondrosarcomas isolated using this method were applied successfully in cDNA microarray experiments. The number of genes that give interpretable results was in the range of what would be expected from microarray results obtained on chondrosarcoma cell line RNA. Signal to noise ratios were good and diVerential expression between tumour and normal cartilage was detectable for a large number of genes. Conclusion—With this newly developed isolation method, high quality RNA can be obtained from low cellular tissue with a high extracellular matrix component. These procedures can also be applied to other tumour material. (J Clin Pathol 2001;54:778‐782)