Guidelines for the use of cell lines in biomedical research.

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

Real-world EGFR testing practices for non-small-cell lung cancer by thoracic pathology laboratories across Europe.

BACKGROUND: Testing for epidermal growth factor receptor (EGFR) mutations is an essential recommendation in guidelines for metastatic non-squamous non-small-cell lung cancer, and is considered mandatory in European countries. However, in practice, challenges are often faced when carrying out routine biomarker testing, including access to testing, inadequate tissue samples and long turnaround times (TATs). MATERIALS AND METHODS: To evaluate the real-world EGFR testing practices of European pathology laboratories, an online survey was set up and validated by the Pulmonary Pathology Working Group of the European Society of Pathology and distributed to 64 expert testing laboratories. The retrospective survey focussed on laboratory organisation and daily EGFR testing practice of pathologists and molecular biologists between 2018 and 2021. RESULTS: TATs varied greatly both between and within countries. These discrepancies may be partly due to reflex testing practices, as 20.8% of laboratories carried out EGFR testing only at the request of the clinician. Many laboratories across Europe still favour single-test sequencing as a primary method of EGFR mutation identification; 32.7% indicated that they only used targeted techniques and 45.1% used single-gene testing followed by next-generation sequencing (NGS), depending on the case. Reported testing rates were consistent over time with no significant decrease in the number of EGFR tests carried out in 2020, despite the increased pressure faced by testing facilities during the COVID-19 pandemic. ISO 15189 accreditation was reported by 42.0% of molecular biology laboratories for single-test sequencing, and by 42.3% for NGS. 92.5% of laboratories indicated they regularly participate in an external quality assessment scheme. CONCLUSIONS: These results highlight the strong heterogeneity of EGFR testing that still occurs within thoracic pathology and molecular biology laboratories across Europe. Even among expert testing facilities there is variability in testing capabilities, TAT, reflex testing practice and laboratory accreditation, stressing the need to harmonise reimbursement technologies and decision-making algorithms in Europe.

Calcium oxalate crystal deposition in the kidney: identification, causes and consequences.

Calcium oxalate (CaOx) crystal deposition within the tubules is often a perplexing finding on renal biopsy of both native and transplanted kidneys. Understanding the underlying causes may help diagnosis and future management. The most frequent cause of CaOx crystal deposition within the kidney is hyperoxaluria. When this is seen in native kidney biopsy, primary hyperoxaluria must be considered and investigated further with biochemical and genetic tests. Secondary hyperoxaluria, for example due to enteric hyperoxaluria following bariatric surgery, ingested ethylene glycol or vitamin C overdose may also cause CaOx deposition in native kidneys. CaOx deposition is a frequent finding in renal transplant biopsy, often as a consequence of acute tubular necrosis and is associated with poorer long-term graft outcomes. CaOx crystal deposition in the renal transplant may also be secondary to any of the causes associated with this phenotype in the native kidney. The pathophysiology underlying CaOx deposition is complex but this histological phenotype may indicate serious underlying pathology and should always warrant further investigation.

Role of Complementary Medicine (Music, Acupuncture, Acupressure, TENS and Audio-Visual Distraction) in Shockwave Lithotripsy (SWL): A Systematic Review From EAU Sections of Urolithiasis (EULIS) and Uro-Technology (ESUT).

We performed a systematic review to look at the role of alternative or complementary medicine such as music, acupressure, acupuncture, transcutaneous electrical nerve stimulation (TENS) and audiovisual distractions to decrease analgesia requirement and alleviate anxiety during SWL. Twenty-three papers(2439 participants) were included: Music (n = 1056.6%), Acupuncture (n = 517.7%), Acupressure (n = 13.8%), TENS (n = 617.2%), and audiovisual distraction (n = 14.6%). Most of the studies showed that complementary therapy, lowered pain, and anxiety with higher patient satisfaction and willingness to undergo the procedure. With its feasibility and convenience, urological guidelines need to endorse it, and more should be done to promote its use in outpatient urological procedures.

Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor.

A human member of the immunoglobulin superfamily was shown to mediate entry of several alphaherpesviruses, including herpes simplex viruses (HSV) 1 and 2, porcine pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1). This membrane glycoprotein is poliovirus receptor-related protein 1 (Prr1), designated here as HveC. Incubation of HSV-1 with a secreted form of HveC inhibited subsequent infection of a variety of cell lines, suggesting that HveC interacts directly with the virus. Poliovirus receptor (Pvr) itself mediated entry of PRV and BHV-1 but not of the HSV strains tested. HveC was expressed in human cells of epithelial and neuronal origin; it is the prime candidate for the coreceptor that allows both HSV-1 and HSV-2 to infect epithelial cells on mucosal surfaces and spread to cells of the nervous system.

Colorectal Serrated Neoplasia: An Institutional 12-Year Review Highlights the Impact of a Screening Programme.

BACKGROUND: As the malignant potential of sessile serrated lesions/polyps (SSL/Ps) and traditional serrated adenomas (TSAs) has been clearly demonstrated, it is important that serrated polyps are identified and correctly classified histologically. AIM: Our aim was to characterize the clinicopathological features of a series of SSL/Ps & TSAs, to assess the accuracy of the pathological diagnosis, the incidence, and the rate of dysplasia in SSL/Ps & TSAs. METHODS: We identified all colorectal serrated polyps between 01/01/2004 and 31/05/2016, by searching the laboratory information system for all cases assigned a "serrated adenoma" SNOMED code. All available and suitable slides were reviewed by one pathologist, who was blinded to the original diagnosis and the site of the polyp. Subsequently discordant cases, SSL/Ps with dysplasia, and all TSAs were reviewed by a second pathologist. RESULTS: Over a 149-month period, 759 "serrated adenoma" polyps were identified, with 664 (from 523 patients) available for review. 41.1% were reviewed by both pathologists; 15.1% (100/664) were reclassified, with the majority being changed from SSL/P to hyperplastic polyp (HYP) (66/664; 9.9%). 80.3% of these HYPs were located in the left colon, and the majority exhibited prolapse effect. There were 520 SSL/Ps (92.2%) & 40 TSAs (7.1%). The majority of SSL/Ps were in the right colon (86.7%) and were small (64.5% <1 cm), while most TSAs were in the left colon (85.7%) and were large (73.1%≥1 cm). 6.7% of SSL/Ps exhibited dysplasia, the majority of which were large (66.7%≥1 cm). Following consensus review, 13/520 (2.5%) SSL/Ps were downgraded from SSL/P with dysplasia to SSL/P without dysplasia. Detection of SSL/Ps peaked in the most recent years reviewed (87.5% reported between 2013 and 2016, inclusive), coinciding with the introduction of "BowelScreen" (the Irish FIT-based colorectal cancer screening programme). CONCLUSIONS: Awareness of, and adherence to, diagnostic criteria is essential for accurate classification of colorectal polyps.

Effects of CDX2 on prognosis and chemotherapy responsiveness in mismatch repair-deficient colorectal cancer.

BACKGROUND: Caudal-related homeobox transcription factor 2 (CDX2) is an intestine-specific transcription factor implicated in tumour differentiation, proliferation, cell adhesion and migration. Negative CDX2 status (CDX2-) is associated with worse prognosis in colorectal cancer and may identify high-risk stage II disease that benefits from adjuvant chemotherapy. This observational study investigated whether CDX2- is associated with prognosis or response to chemotherapy in the mismatch repair-deficient (dMMR) phenotype of colorectal cancer. METHODS: Patients with resectable dMMR colorectal cancer were eligible for inclusion. The prognostic and predictive value of CDX2 expression on the presence of lymph node metastasis (LNM) and survival was investigated. CDX2 status was determined via immunohistochemistry using the Leica Bond™ CDX2 (clone EP25) ready-to-use primary antibody. RESULTS: Some 235 of 238 consecutive dMMR tumours were assessed for CDX2 status. CDX2- was observed in 15·7 per cent of colorectal cancer. Interobserver agreement was excellent (κ = 0·863; P < 0·001). CDX2- was significantly associated with female sex, increased size, advanced stage, worse conventional and poorly differentiated cluster (PDC) grade, mucinous morphology, perineural and lymphovascular invasion, and pN status (all P ≤ 0·038). CDX2- was not associated with LNM or survival in multivariable analysis. Independent predictors of LNM were PDC grade (odds ratio (OR) 4·12, 95 per cent c.i. 1·76 to 9·63; P = 0·001) and extramural venous invasion (OR 3·79, 1·62 to 8·85; P = 0·002). Budding (hazard ratio (HR) 2·79, 95 per cent c.i. 1·60 to 4·87; P < 0·001), pT status (HR 3·59, 1·29 to 10·01; P = 0·015) and adjuvant chemotherapy (HR 2·07, 1·15 to 3·74; P = 0·016) were independently associated with worse disease-free survival. CONCLUSION: CDX2- does not confer a worse prognosis in the dMMR phenotype of colorectal cancer. The MMR status of patients with colorectal cancer should be determined before assessing CDX2 status.

Effective removal of equine arteritis virus from stallion semen.

REASONS FOR PERFORMING STUDY: A method of removing equine arteritis virus (EAV) from equine semen used for artificial insemination is urgently needed. Recent medical studies suggest that a double semen processing technique of density gradient centrifugation followed by a 'swim-up' can provide virus-free sperm preparations for assisted reproduction. OBJECTIVES: To investigate the use of the double semen processing technique to obtain virus-free sperm preparations from stallion semen containing EAV. METHODS: Aliquots of an ejaculate from an uninfected stallion were spiked with virus and processed by the double processing technique. The sperm preparations were tested by PCR for the presence of EAV. The procedure was repeated using an ejaculate from a known shedding stallion, testing processed and unprocessed aliquots by PCR and virus isolation. RESULTS: Virus-free sperm preparations were obtained using the double sperm processing technique. The 'swim-up' step is apparently required to ensure complete virus removal. CONCLUSIONS: The double semen processing technique is potentially a useful and simple tool for the removal of EAV from the semen of shedding stallions. POTENTIAL RELEVANCE: The inclusion of density gradient centrifugation and 'swim-up' in protocols for the processing of semen for artificial insemination could help prevent the transmission of viral diseases carried in semen, such as EAV.

Microsatellite instability is associated with reduced disease specific survival in stage III colon cancer.

BACKGROUND: Up to 15% of colorectal cancers exhibit microsatellite instability (MSI), where errors in replication go unchecked due to defects in the mismatch repair system. This study aimed to determine survival in a large single-centre series of 1250 consecutive colorectal cancers subjected to universal MSI testing. METHODS: Clinical and pathological features of patients with colorectal cancer identified on prospectively maintained colorectal and pathology databases at St. Vincent's University Hospital from 2004 to May 2012 were examined. Mismatch repair (MMR) status was determined by immunohistochemistry. Kaplan-Meier curves, the log-rank test and Cox regression were used to associate survival with clinical and pathological characteristics. RESULTS: Of the 1250 colorectal cancers in the study period, 11% exhibited MSI (n = 138). Patients with MSI tumours had significantly lower rates of lymph node and distant metastases (MSI N+ rate: 24.8% compared with MSS N+ rate: 46.2%, p < 0.001). For Stage I and II disease MSI was associated with improved disease free survival (DSS) compared with MSS colon cancer. However, patients with Stage III MSI colon cancers had a worse DSS than those with MSS tumours. Stage III MSI tumours exhibited higher rates of lymphovascular invasion and perineural invasion than Stage I/II MSI tumours. CONCLUSION: MSI is associated with a reduced risk of nodal and distant metastases, with an improved DSS in Stage I/II colon cancer. However, when MSI tumours progress to Stage III these patients had worse outcomes and pathological features. New strategies for this cohort of patients may be required to improve outcomes.

Premedication for bronchoscopy in older patients: a double-blind comparison of two regimens.

INTRODUCTION: Older patients are the most prevalent age cohort requiring bronchoscopy. Prior sedation should be offered to improve patient comfort and operator technical ease. Older patients have increased sensitivity to centrally acting drugs increasing the procedural risk. This perceived risk may limit access to bronchoscopy in older patients. There have been no systematic prospective placebo-controlled studies in older patients. We compared a novel premedication regimen-oral temazepam plus nebulised Lignocaine (new treatment) to an established regimen of intravenous alfentanyl (control). METHODS: Consecutive patients 75 years and older referred for bronchoscopy were considered. Twenty-five patients were randomly assigned to each group. The primary outcome measure was the lowest oxygen saturation recorded from the administration of IV drugs and for 30 min post-bronchoscopy. RESULTS: The lowest mean oxygen saturation in the new treatment group was 92.2% (90.3-94.2) and in the control group 91.1% (89.2-93.1). This was not statistically different (P = 0.370). There were no adverse events. CONCLUSION: This is the largest prospective study to date on an older population undergoing bronchoscopy supporting previous retrospective findings regarding the safety of this procedure. Determined by oxygen saturations there is no difference in safety between premedication regimens comprising oral temazepam/nebulised lignocaine or intravenous alfentanyl.

Organization of the rat Tage4 gene and herpesvirus entry activity of the encoded protein.

The Tage4 gene (Tumor-Associated Glycoprotein E4) is a member of the immunoglobulin superfamily overexpressed in rat colon tumors and Min mouse intestinal adenomas. The Tage4 cDNA presents approximately 60% identity with the human CD155, a member of the immunoglobulin superfamily coding for a transmembrane protein capable of serving as an entry receptor for poliovirus, porcine pseudorabies virus and bovine herpesvirus 1. We determined the structure of the Tage4 gene. This gene covers approximately 15 kb and is composed of eight exons and seven introns. We also isolated approximately 2 kb of the 5' flanking region of the Tage4 gene and demonstrated the existence of closely clustered transcription start sites. No splicing variant was identified by RT-PCR indicating that the Tage4 gene is transcribed as a unique mRNA. Finally, the protein encoded by the Tage4 gene was tested for ability to mediate entry of several viruses. These structural and functional features of the rat Tage4 gene were compared to those of the human CD155 gene. The results indicated that the Tage4 gene is probably orthologous to the gene for CD155.

Contributions of gD receptors and glycosaminoglycan sulfation to cell fusion mediated by herpes simplex virus 1.

Two cell surface proteins (nectin-1/HveC and nectin-2/HveB) shown previously to serve as receptors for the entry of herpes simplex virus 1 (HSV-1) wild-type and/or mutant strains were found to serve also as receptors for HSV-1-induced cell fusion. Transfection with genomic DNA from a syncytial HSV-1 strain encoding wild-type gD resulted in fusion of Chinese hamster ovary (CHO) cells expressing nectin-1 but not of cells expressing nectin-2. In contrast, transfection with DNA from a related HSV-1 strain encoding the mutant Rid1 form of gD resulted in fusion of CHO cells expressing either receptor but not of control cells. These results are consistent with the ability of each receptor to mediate entry of viruses expressing wild-type or Rid1 gD and with results obtained previously with HVEM (HveA), a third HSV-l entry receptor. Undersulfation of GAGs in receptor-expressing cell lines predictably reduced susceptibility to HSV-l infection. In contrast, susceptibility to cell fusion mediated by HVEM or nectin-1 was not reduced. Undersulfation of GAGs partially inhibited cell fusion mediated by nectin-2. We conclude that HSV-1-induced cell fusion requires a gD-binding entry receptor, that ability of an HSV-1 strain to use HVEM, nectin-2 or nectin-1 for cell fusion depends on the allele of gD expressed and that GAGs may influence cell fusion, dependent on the gD-binding receptor used, but are less important for cell fusion mediated by HVEM, nectin-2 or nectin-l than for viral entry.

Internal quality assurance in a clinical virology laboratory. II. Internal quality control.

AIMS: In April 1991 additional quality control procedures were introduced into the virology section of the Clinical Microbiology and Public Health Laboratory, Cambridge. Internal quality control (IQC) samples were gradually included in the serological assays performed in the laboratory and supplemented kit controls and standard sera. METHODS: From April 1991 to December 1993, 2421 IQC procedures were carried out with reference sera. RESULTS: The IQC samples were evaluated according to the Westgard rules. Violations were recorded in 60 of 1808 (3.3%) controls and were highest in the IQC samples of complement fixation tests (25/312 (8%) of controls submitted for complement fixation tests). CONCLUSIONS: The inclusion of IQC samples in the serological assays performed in the laboratory has highlighted batch to batch variation in commercial assays. The setting of acceptable limits for the IQC samples has increased confidence in the validity of assay results.

Internal quality assurance in a clinical virology laboratory. I. Internal quality assessment.

AIMS: In April 1991 an internal quality assessment scheme (IQAS) was introduced into the virology section of the Clinical Microbiology and Public Health Laboratory, Cambridge. The IQAS was established to identify recurring technical and procedural problems, to check the adequacy of current techniques, and to calculate the frequency of errors. METHODS: Between April 1991 and December 1993, 715 anonymous clinical serum samples were submitted to the laboratory to test 3245 individual procedures of diagnostic viral serology. RESULTS: A total of 485 (14.9%) procedural and 61 (1.9%) technical discrepancies were observed, the technical discrepancies mainly being recorded in complement fixation tests. Twenty two (0.7% of total procedures) of the technical discrepancies were diagnostically significant. CONCLUSIONS: Evaluation criteria developed with the introduction of IQAS to viral serology, and technical and procedural discrepancies are assessed. As yet, IQAS has not been introduced to other sections of the diagnostic virology laboratory (virus isolation, electron microscopy, immunofluorescence, and enzyme linked immunosorbent assays for viral and chlamydial antigens).

Biological activity and receptor binding properties of some analogues of pQDPFLRFamide.

To investigate the role of the N-terminal region of the heptapeptide FMRFamide-like peptide, pQDPFLRFamide, three analogues were synthesized. The analogues [pQNPFLRFamide, pQDAibFLRFamide (Aib = aminoisobutyric acid) and pQDGFLRFamide] contained modifications at amino acid residues 2 and 3, which we believed might be critical for maintaining the bioactive conformation of the heptapeptide. The analogues were tested for their ability to bind to receptors in membranes from Helix aspersa circumoesophageal ganglia and for their biological effects on the isolated Helix heart, the Helix tentacle retractor muscle, and extensor-tibiae neuromuscular preparation of the locust. Schistocerca gregaria. The substitution of Asn for Asp2 and that of Aib for Pro3 were conservative with respect to retention of heptapeptide-like biological activity, whereas the substitution of Gly for Pro3 significantly improved the binding affinity of the peptide for the FMRFamide receptors and conferred on the peptide some characteristic FMRFamide-like biological activity. Thus, pQDPFLRFamide bioactivity may depend on a bent conformation in solution.

Biological activity and receptor binding properties of some C-terminally modified analogues of FMRFamide.

The functional role of the C-terminal amide group (-CONH2) of the molluscan regulatory peptide FMRFamide has been examined in two sets of analogues based on FnLKFamide and FnLRFamide (nL = norleucine). In each series the amide group was replaced by -CONHCH3, -CON(CH3)2, -CONHNH2, -COOCH3, -CH2OH, and -COOH. The analogues were tested for their ability to bind to receptors in membranes from Helix aspersa circumoesophageal ganglia and for their biological effects on the isolated Helix heart. The results indicate i) that agonist activity, but not binding to the receptor, requires the presence of the amide carbonyl group; ii) the hydrogen atoms of the amide group are not essential either for binding or for agonist activity (the mono- and dimethylamides were more effective than the parent compounds on both counts); iii) the is more effective an agonist than is the amide in stimulating Helix heart.

Bronchodilator response to nebulized salbutamol in elderly patients with stable chronic airflow limitation.

The bronchodilator response to 5 mg nebulized salbutamol was studied in 20 elderly patients with stable chronic airflow limitation. Salbutamol produced significantly greater increases in FEV1 and FVC compared with placebo although there was no difference in subjective sensation of breathlessness. Spirometry can be successfully used to assess respiratory function in appropriately selected elderly patients with chronic airflow limitation.

Chronic eosinophilic pneumonia. Diagnosis by fine needle aspiration.

A case of chronic eosinophilic pneumonia in which the diagnosis was established by fine needle aspiration of the pulmonary lesions is presented. The cytologic findings included the presence of an inflammatory exudate consisting of 40% eosinophils, 25% neutrophils and the remaining cells comprised of lymphocytes, histiocytes, atypical columnar cells and cells probably of pneumocyte type II origin. Fine needle aspiration of pulmonary infiltrates can be quite helpful in confirming the diagnosis of chronic eosinophilic pneumonia and in differentiating it from other similar conditions including malignant lymphomas, if the clinical and radiologic appearances are also taken into consideration.