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In ruminants Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of a chronic granulomatous inflammatory bowel disease called Johne's disease (JD). Some researchers have hypothesised that MAP is also associated with Crohn's disease (CD), an inflammatory bowel disease in humans that shares some histological features of JD. Despite numerous attempts to demonstrate causality by researchers, direct microbiological evidence of MAP involvement in CD remains elusive. Importantly, it has not been possible to reliably and reproducibly demonstrate mycobacteria in the tissue of CD patients. Past attempts to visualise mycobacteria in tissue may have been hampered by the use of stains optimised for Mycobacterium tuberculosis complex (MTB) and the lack of reliable bacteriological culture media for both non-tuberculous mycobacteria (NTM) and cell-wall-deficient mycobacteria (CWDM). Here we describe a Ziehl-Neelsen (ZN) staining method for the demonstration of CWDM in resected tissue from patients with Crohn's disease, revealing the association of CWDM in situ with host tissue reactions, and posit this as a cause of the tissue inflammation. Using the ZN stain described we demonstrated the presence of CWDM in 18 out of 18 excised tissue samples from patients diagnosed as having Crohn's disease, and in zero samples out of 15 non-inflammatory bowel disease controls.

Traditionally, cancer diagnosis and management has been reactionary in that symptoms lead to investigations, then a diagnosis is followed by clinical management. This process is heavily dependent on tissue diagnosis mainly by histopathology and to a lesser extent, cytopathology. However, in recent times there has been a shift towards precision medicine to enable prevention, prediction and personalisation in healthcare. The core of precision medicine is optimising therapeutic benefit for patients, by using genomic and molecular profiling, analogously termed precision pathology. This review explores (1) the evolution of pathology from a para-clinical discipline to a mainstream medical field integral to oncology tumour boards; (2) its critical role in preventative, diagnostic, therapeutic and follow-up cancer care; (3) the future of tissue pathology in the era of precision oncology; and (4) how pathologists may evolve to future-proof their profession.

Sir,Hypophosphatasia is an inborn error of metabolism that arises from mutations in the TNSALP gene located on chromosome 1p36.1–p34 (MIM# 171760). The common defect in all types of hypophosphatasi...

Aims To investigate the association between GFR and body composition in healthy young men with differing body mass. Methods Three age-matched groups were recruited: normal BMI (n = 22) 30 kg/m2 and BIA body fat ≤20%, and obese (n = 22) with BMI ≥30 kg/m2 and BIA body fat > 30%. Dietary analyses, GFR by clearance of 99mTc-DTPA, and body composition by dual-energy X-ray absorptiometry (DEXA) were measured in all participants. Results Muscular men had higher GFR (mean 186.4 mL/min; 95% CI 171.7–201.1) than normal BMI and obese groups (p = 0.0007). Fat mass protein intake, and smoking status were not associated with GFR; whereas lean mass had the strongest association with GFR. In all subgroups, skeletal muscle mass correlated significantly with GFR (p = 0.04). In multi-variate models, variables with the strongest associations with GFR were age (p = 0.0009) and lean mass (p = 0.0001). Conclusions Age and lean mass were strong determinants of GFR in healthy men of various body compositions. We estimate that GFR decreases by 1 mL/min/year of age and increases 2.3 mL/min/kg of lean mass in healthy men.

Background and aims Familial dysalbuminaemic hyperthyroxinaemia (FDH) is an important cause of discordant thyroid function tests (TFT). Free thyroxine (T4) levels are falsely elevated on some assays and current diagnostic tests for FDH are laborious. We aimed to investigate a novel, mass spectrometry approach to diagnosis of FDH in a 51-year-old man with persistently elevated free T4 levels, discordant normal TSH and normal free triiodothyronine. He was clinically euthyroid and had a daughter with similar TFT profile. Methods Serum albumin was assessed using electrospray time-of-flight (TOF) liquid chromatography/mass spectrometry (LC/MS). Targeted sequencing of exons 3 and 7 of the albumin gene was used to confirm mutations and to screen 15 archived DNA samples that were negative for thyroid hormone receptor mutations. Results LC/MS demonstrated heterozygosity for an albumin variant with a 19 Da decrease in mass, indicative of an Arg→His substitution. This was confirmed by DNA sequencing which showed a guanine to adenine transition at codon 218 of exon 7, a mutation known to increase T4 binding. Discussion TOF LC/MS is a novel procedure for diagnosing FDH. The test is rapid (taking

Background: Trace elements are commonly measured by inductively coupled mass spectrometry (ICP-MS). A 30-year-old man had a plasma selenium (Se) concentration on ICP-MS of 66 µmol/L (reference interval 0.45–1.40), a potentially lethal level, despite no history of Se exposure or toxicity symptoms. He had earlier undergone magnetic resonance imaging with a gadolinium (Gd) contrast agent, which is known to interfere with Se on ICP-MS. We aimed to adjust our method by monitoring a second Se isotope that is unaffected by Gd to detect this preanalytical interference. Methods: Plasma samples referred for trace metal testing had Se measured on ICP-MS (monitoring 78Se), which we modified to also monitor a second isotope (82Se). The modified method was then applied to a specimen with known Gd contamination. Results: Plasma Se results ( n = 41) derived from monitoring the two different Se isotopes were similar with a good correlation ( R2 = 0.991) over a range of 0.23–2.21 µmol/L. On repeat analysis, our patient had a Se concentration of 65 µmol/L using the 78Se isotope but only 1.43 µmol/L using 82Se. Conclusion: To avoid reporting a falsely elevated plasma Se result, we suggest that Se analysis by ICP-MS should include a second Se isotope for monitoring, that is not subject to Gd interference.

Background Clinical guidelines recommend reporting estimated glomerular filtration rate (eGFR) based on plasma creatinine to predict kidney function. This study aimed to assess whether inclusion of cystatin C could improve accuracy of prediction equations in comparison to a gold standard in cancer patients and a healthy normal group. Methods Plasma creatinine and cystatin C was analysed in 155 patients (cancer, n = 80; kidney donors, n= 75) undergoing Tech-netium-DTPA GFR procedure. The performance of three equations by the CKD-EPI group (creatinine-, creatinine and cystatin C-, cystatin C-based) was compared to Tc-DTPA GFR. Results Overall, the combination of creatinine and cystatin C equation had the smallest GFR underestimation of -5.7 mL/min/ 1.73 m 2 (95%CI -8.9 to -2.4). Diagnostic sensitivity and specificity was highest for this equation in the overall population (AUC = 0.855, p 0.05), however the proportion of results for all equations within 30% of GFR was not different for the overall population, nor in subgroup analyses. Conclusion: The inclusion of cystatin C together with creatinine in prediction equations marginally improved the prediction of GFR in the overall population and in males. More research is required to improve the accuracy of prediction equations in cancer patients for guiding therapy.

Aims To compare estimated glomerular filtration rate (eGFR) according to the CKD-EPI equation, with (CKD-EPI, mL/min/1.73m2) and without body surface area (BSA)-normalisation (CKD-EPI_;BSA, mL/min), against measured Tc-DTPA GFR (mL/min). Methods The CKD-EPI and CKD-EPI_;BSA equations were compared in 222 individuals with Tc-DTPA GFR for bias, proportion within 30% of GFR (P30) and area under the receiver-operator curve (ROC) for detecting GFR Results Chi-square indicated differences in P30 s between CKD-EPI_;BSA and CKD-EPI: in those with BMI ≥30 kg/m2 (32%), in those with BMI > 25.0–29.9 kg/m2 (18%) and in those with BMI ≥18.5–25.0 kg/m2 (2%) (p 125 mL/min (0.81) was greater than for the CKD-EPI (0.80 and 0.71, respectively). Concordance for carboplatin dosing using the CKD-EPI_;BSA equation was 71% and 56.% by the CKD-EPI equation (p = 0.07). Conclusion Estimation of absolute Tc-DTPA GFR using the CKD-EPI equation was improved by removal of BSA normalisation, reflected by higher proportion of results within 30% of GFR and less underestimations of GFR, with implications for drug dosing.

Introduction The partial pressure of oxygen at half saturation (p50) is useful to detect altered oxygen-affinity states in isolated polycythaemia. Commercial gas analysers calculate this parameter from a single oxygen tension (PO 2 ) and saturation (sO 2 ) measurement utilising the Hill plot. The accuracy of these calculated p50s in patients with variant haemoglobin is not well documented. We compared results from two commercial analysers (Radiometer ABLFlex 800 and Siemens Rapidlab 1265) with tonometry for five patients with variant haemoglobin or red-cell enzyme deficiency and one control subject. Methods A reference interval for p50 was determined. Venous blood was analysed sequentially on the commercial analysers and by tonometry. Hill plots were prepared from the tonometry data and the Hill coefficient ( n ) derived, least-squares regression was used to calculate the standard error. Results The reference interval was 22–27mmHg. The p50 values showed good agreement between analysers, and between analysers and tonometry, except in the case of Haemoglobin Heathrow. Discussion Measuring p50 on a commercial gas analyser is convenient, readily available and shows good agreement with tonometry, except in haemoglobin variants with altered cooperativity ( n ), as in the case of Haemoglobin Heathrow. Gas-analyser derived numerical estimates of p50 for haemoglobin variants with altered cooperativity should be interpreted with caution; in this situation formal tonometry is more accurate.

Aims Haemolysis is an important source of assay interference, especially in the Emergency Department (ED). A rapid-clotting serum tube, Becton-Dickinson (BD) Vacutainer Rapid Serum Tube (RST), was reported to reduce haemolysis rates compared to standard serum tubes. We evaluated the effect of RST on haemolysis markers compared with our current STAT testing tube, BD Vacutainer Plasma Separator Tube (PST). Methods Patients presenting to the ED over a 8-day period had an additional RST and BD Vacutainer non-gel heparin plasma tube collected. Blood was collected from 347 patients by trained nursing staff from an intravenous cannula using an adaptor device (Vacutainer Luer-Lok access device). All samples were then analysed for six haemolysis markers: haemolysis index, phosphate, magnesium, potassium, lactate dehydrogenase (LD), and aspartate aminotras-ferase (AST). Results We found 9% of samples were haemolysed (>0.5g/L free haemoglobin). Significant differences (p p 0.05) higher for phosphate and potassium and lower for LD. Discussion/conclusion RST tubes did not improve markers of haemolysis compared to plasma tubes.

Summary Metallothioneins (MT) are low molecular weight, metal-binding proteins. The induction of MT synthesis by cytokines, hormones and other cytotoxic agents indicates its role in cellular proliferation and differentiation as well as in cellular defense mechanisms. Previous studies have detected expression of MT in various human tumors by immunohistochemical staining. In certain cases the presence of MT in a tumor may be associated with its resistance against radiation and chemotherapeutic agents. Immunohistochemical staining of MT using a rabbit polyclonal anti-rat liver MT antibody was carried out in eight gastric, two small bowel and one large bowel lymphomas, and in ten control gastrointestinal (gastric, colonic and small bowel) biopsies or excised bowel segments with benign lymphoid infiltrates. The primary antibody against rat liver MT readily cross-reacts with human MT. The neoplastic cells in nine of 11 malignant lymphomas showed weak to intense staining for MT, mostly in cytoplasm. In these cells a few nuclei (less than 5% of all tumor cells) were stained positively for MT. The benign lymphocytes in the gastrointestinal excised specimens and biopsies were mostly negative; four cases showed few positive cells. The giant cells were also positive for MT in a Crohn’s disease case. We conclude that the presence or absence of MT in lymphocytes, detected by immunohistochemistry, may indicate the growth patterns of these cells. The distinct pattern of MT staining in malignant lymphoma in our study is suggestive of a potential use of MT staining as a discriminator between benign and malignant lymphoid tissues.

A genetic variant of antithrombin with impaired heparin cofactor activity was identified in 4 members of a French family. Both the variant and normal antithrombin component were purified by affinity chromatography on heparin Sepharose. Reverse phase peptide mapping revealed a single altered peak when tryptic digests of both antithrombins were compared. After further purification of the aberrant peptide, amino acid analysis indicated a substitution of 41 Leu----Pro (antithrombin Basel). This result was confirmed by liquid secondary ion mass spectrometry which gave a measured mass of 816.4655 Da for the new peptide compared to a calculated mass of 800.3579 Da for the normal peptide and 816.4579 for the Leu----Pro substitution.

The distribution of metallothionein (MT) during human development was investigated using both immunohistochemical and biochemical methods. The level of MT in the fetal liver was higher than the adult liver levels. Higher levels of zinc (Zn) and copper (Cu) were also detected in the fetal liver compared to the adult liver. Although cadmium (Cd) was present in detectable levels in the human adult liver, none was detected in the human fetal liver. MT was localized in the nucleus and the cytoplasm of human fetal and neonatal hepatocytes, using a specific rabbit antibody raised to rat liver MT. In the adult human liver cells, MT was localized mainly in the cytoplasm. In the fetal and neonatal human kidney, MT was localized mainly in the nucleus and the cytoplasm of the proximal tubular epithelial cells. In the adult kidney, in addition to nuclear-cytoplasmic localization of MT, intraluminal localization was also observed.