openFrame: A modular, sustainable, open microscopy platform with single-shot, dual-axis optical autofocus module providing high precision and long range of operation.

'openFrame' is a modular, low-cost, open-hardware microscopy platform that can be configured or adapted to most light microscopy techniques and is easily upgradeable or expandable to multiple modalities. The ability to freely mix and interchange both open-source and proprietary hardware components or software enables low-cost, yet research-grade instruments to be assembled and maintained. It also enables rapid prototyping of advanced or novel microscope systems. For long-term time-lapse image data acquisition, slide-scanning or high content analysis, we have developed a novel optical autofocus incorporating orthogonal cylindrical optics to provide robust single-shot closed-loop focus lock, which we have demonstrated to accommodate defocus up to ±37 µm with <200 nm accuracy, and a two-step autofocus mode which we have shown can operate with defocus up to ±68 µm. We have used this to implement automated single molecule localisation microscopy (SMLM) in a relatively low-cost openFrame-based instrument using multimode diode lasers for excitation and cooled CMOS cameras.

Technical considerations when designing a gene expression panel for renal transplant diagnosis.

Gene expression analysis is emerging as a new diagnostic tool in transplant pathology, in particular for the diagnosis of antibody-mediated rejection. Diagnostic gene expression panels are defined on the basis of their pathophysiological relevance, but also need to be tested for their robustness across different preservatives and analysis platforms. The aim of this study is the investigate the effect of tissue sampling and preservation on candidate genes included in a renal transplant diagnostic panel. Using the NanoString platform, we compared the expression of 219 genes in 51 samples, split for formalin-fixation and paraffin-embedding (FFPE) and RNAlater preservation (RNAlater). We found that overall, gene expression significantly correlated between FFPE and RNAlater samples. However, at the individual gene level, 46 of the 219 genes did not correlate across the 51 matched FFPE and RNAlater samples. Comparing gene expression results using NanoString and qRT-PCR for 18 genes in the same pool of RNA (RNAlater), we found a significant correlation in 17/18 genes. Our study indicates that, in samples from the same routine diagnostic renal transplant biopsy procedure split for FFPE and RNAlater, 21% of 219 genes of potential biological significance do not correlate in expression. Whether this is due to fixatives or tissue sampling, selection of gene panels for routine diagnosis should take this information into consideration.

IgG4-related disease in a multi-ethnic community: clinical characteristics and association with malignancy.

BACKGROUND: Immunoglobulin-G4-related disease (IgG4-RD) is a recently recognized fibro-inflammatory condition that can affect multiple organs. Despite growing interest in this condition, the natural history and management of IgG4-RD remain poorly understood. AIM: To describe the clinical characteristics, treatment and outcomes of IgG4-RD in a multi-ethnic UK cohort, and investigate its possible association with malignancy. DESIGN: Retrospective analysis of case-note and electronic data. METHODS: Cases were identified from sub-specialty cohorts and a systematic search of an NHS trust histopathology database using 'IgG4' or 'inflammatory pseudotumour' as search terms. Electronic records, imaging and histopathology reports were reviewed. RESULTS: In total, 66 identified cases of IgG4-RD showed a similar multi-ethnic spread to the local population of North West London. The median age was 59 years and 71% of patients were male. Presenting symptoms relating to mass effect of a lesion were present in 48% of cases and the mean number of organs involved was 2.4. Total of 10 patients had reported malignancies with 6 of these being haematological. 83% of those treated with steroids had good initial response; however, 50% had relapsing-remitting disease. Rituximab was administered in 11 cases and all achieved an initial serological response. Despite this, seven patients subsequently relapsed after a mean duration of 11 months and four progressed despite treatment. CONCLUSIONS: We report a large UK-based cohort of IgG4-RD that shows no clear ethnic predisposition and a wide range of affected organs. We discuss the use of serum IgG4 concentrations as a disease marker in IgG4-RD, the association with malignant disease and outcomes according to differing treatment regimens.

The Banff 2017 Kidney Meeting Report: Revised diagnostic criteria for chronic active T cell-mediated rejection, antibody-mediated rejection, and prospects for integrative endpoints for next-generation clinical trials.

The kidney sessions of the 2017 Banff Conference focused on 2 areas: clinical implications of inflammation in areas of interstitial fibrosis and tubular atrophy (i-IFTA) and its relationship to T cell-mediated rejection (TCMR), and the continued evolution of molecular diagnostics, particularly in the diagnosis of antibody-mediated rejection (ABMR). In confirmation of previous studies, it was independently demonstrated by 2 groups that i-IFTA is associated with reduced graft survival. Furthermore, these groups presented that i-IFTA, particularly when involving >25% of sclerotic cortex in association with tubulitis, is often a sequela of acute TCMR in association with underimmunosuppression. The classification was thus revised to include moderate i-IFTA plus moderate or severe tubulitis as diagnostic of chronic active TCMR. Other studies demonstrated that certain molecular classifiers improve diagnosis of ABMR beyond what is possible with histology, C4d, and detection of donor-specific antibodies (DSAs) and that both C4d and validated molecular assays can serve as potential alternatives and/or complements to DSAs in the diagnosis of ABMR. The Banff ABMR criteria are thus updated to include these alternatives. Finally, the present report paves the way for the Banff scheme to be part of an integrative approach for defining surrogate endpoints in next-generation clinical trials.

The Banff 2015 Kidney Meeting Report: Current Challenges in Rejection Classification and Prospects for Adopting Molecular Pathology.

The XIII Banff meeting, held in conjunction the Canadian Society of Transplantation in Vancouver, Canada, reviewed the clinical impact of updates of C4d-negative antibody-mediated rejection (ABMR) from the 2013 meeting, reports from active Banff Working Groups, the relationships of donor-specific antibody tests (anti-HLA and non-HLA) with transplant histopathology, and questions of molecular transplant diagnostics. The use of transcriptome gene sets, their resultant diagnostic classifiers, or common key genes to supplement the diagnosis and classification of rejection requires further consensus agreement and validation in biopsies. Newly introduced concepts include the i-IFTA score, comprising inflammation within areas of fibrosis and atrophy and acceptance of transplant arteriolopathy within the descriptions of chronic active T cell-mediated rejection (TCMR) or chronic ABMR. The pattern of mixed TCMR and ABMR was increasingly recognized. This report also includes improved definitions of TCMR and ABMR in pancreas transplants with specification of vascular lesions and prospects for defining a vascularized composite allograft rejection classification. The goal of the Banff process is ongoing integration of advances in histologic, serologic, and molecular diagnostic techniques to produce a consensus-based reporting system that offers precise composite scores, accurate routine diagnostics, and applicability to next-generation clinical trials.

Banff Borderline Changes Suspicious for Acute T Cell-Mediated Rejection: Where Do We Stand?

The definition of Banff Borderline became ambiguous when the Banff 2005 consensus modified the lower threshold from i1t1 (10-25% interstitial inflammation with mild tubulitis) to i0t1 (0-10% interstitial inflammation with mild tubulitis). We conducted a worldwide survey among members of the Renal Pathology Society about their approach to this diagnostic category. A web-based survey was sent out to all 503 current members (153 respondents). A database search yielded which threshold for Banff i was applied in the most influential manuscripts about Borderline. Among the 139 nephropathologists using the Borderline category, 67% use the Banff 1997 definition, requiring Banff i1. Thirty-seven percent admitted to sometimes exaggerating Banff i in the presence of tubulitis, to reach a diagnosis of Borderline. Forty-eight percent were dissatisfied with the definition of Borderline. The majority of the most influential manuscripts used the 1997 definition, contrary to the current one. There is considerable dissatisfaction with Borderline, and practice in Banff i thresholds is variable. Until additional studies inform a revision, we suggest leaving it to each pathologist's discretion whether to use i0 or i1 as the minimal threshold. In order to avoid future ambiguity, a web-based synopsis of all scattered current Banff definitions and rules should be created.

Diagnosis of early pancreas graft failure via antibody-mediated rejection: single-center experience with 256 pancreas transplantations.

Early pancreas graft loss is usually attributed to technical failure while the possibility of antibody-mediated rejection (AMR) is generally overlooked. To investigate the role of AMR in early pancreas graft loss, we retrospectively assessed 256 patients with simultaneous pancreas-kidney transplantation (SPK) between 1985 and 2010 at our institute. We included 33 SPK patients who lost their pancreas graft <1 year after transplantation. AMR was diagnosed based on donor-specific antibodies, C4d and histology in 7 cases, 8 cases were suspicious for AMR and 18 pancreas graft losses were not due to AMR. Acute AMR occurred >1 month after transplantation in 6/7 cases, whereas all other causes typically led to loss <1 month after transplantation. Thrombotic lesions occurred equally among the 33 cases. In 12/18 concurrent kidney specimens, the diagnostic results paralleled those of the pancreas graft. All patients with acute AMR of the pancreas graft lost their renal grafts <1 year after transplantation. In the setting of a thrombotic event, histopathological analysis of early pancreas graft loss is advisable to rule out the possibility of AMR, particularly because a diagnosis of acute AMR has important consequences for renal graft outcomes.

Microcirculation inflammation associates with outcome in renal transplant patients with de novo donor-specific antibodies.

In renal transplant patients with de novo donor-specific antibodies (dnDSA) we studied the value of microcirculation inflammation (MI; defined by the addition of glomerulitis (g) and peritubular capillaritis (ptc) scores) to assess long-term graft survival in a retrospective cohort study. Out of all transplant patients with standard immunological risk (n = 638), 79 (12.4%) developed dnDSA and 58/79 (73%) had an indication biopsy at or after dnDSA development. Based on the MI score on that indication biopsy patients were categorized, MI0 (n = 26), MI1 + 2 (n = 21) and MI ≥ 3 (n = 11). The MI groups did not differ significantly pretransplantation, whereas posttransplantation higher MI scores developed more anti-HLA class I + II DSA (p = 0.011), showed more TCMR (p < 0.001) and showed a trend to C4d-positive staining (p = 0.059). Four-year graft survival estimates from time of indication biopsy were MI0 96.1%, MI1 + 2 76.1% and MI ≥ 3 17.1%; resulting in a 24-fold increased risk of graft failure in the MI ≥ 3 compared to the MI0 group (p = 0.003; 95% CI [3.0-196.0]). When adjusted for C4d, MI ≥ 3 still had a 21-fold increased risk of graft failure (p = 0.005; 95% CI [2.5-180.0]), while C4d positivity on indication biopsy lost significance. In renal transplant patients with de novo DSA, microcirculation inflammation, defined by g + ptc, associates with graft survival.

Outcome of patients with preformed donor-specific antibodies following alemtuzumab induction and tacrolimus monotherapy.

It has been shown that low-level preformed donor-specific antibodies (DSAbs) detected by luminex beads in the setting of a negative CDC and flow cytometry crossmatch (CDC/FCXM) are associated with inferior allograft outcomes. The relevance of preformed DSAbs in patients receiving alemtuzumab induction and tacrolimus monotherapy has not been studied. Four hundred and eighty renal transplant recipients with a negative CDC/FCXM had their pretransplant sera retrospectively screened for DSAbs. 45/480 (9.4%) of patients were found to have preformed DSAbs. Females and patients receiving regrafts were more likely to have a DSAb (p = 0.008 and p < 0.0001, respectively). Patients with DSAbs had inferior allograft survival (p = 0.047), increased incidence of antibody-mediated rejection (p < 0.0001) and inferior allograft function at 6 months posttransplant (p = 0.017). Patients with HLA class I DSAb (alone or in combination with a Class II DSAb) with high mean fluorescence intensities (MFIs) were at highest risk. We conclude that patients with preformed DSAb are at high risk of adverse outcomes when receiving a minimal immunosuppressive regime incorporating alemtuzumab induction. Patients found to have a preformed DSAb despite a negative crossmatch might benefit from augmented immunosuppression.

Stem cells and renal regeneration.

The role of embryonal or adult stem cells, in particular bone marrow (BM)-derived stem cells, in regenerating the kidney after injury has been the subject of intensive investigation. BM-derived stem cells have been shown to give rise to small numbers of most renal cell types, including tubular cells, mesangial cells, podocytes, vascular cells and interstitial cells. However, the role this infrequent display of BM cell plasticity plays in organ regeneration is less certain. Injections of BM-derived cells do improve renal function in many animal models of renal disease. Current opinion attributes this renoprotective effect mainly to paracrine factors supporting regeneration by local renal cells and to immunomodulatory effects, rather than to transdifferentiation of BM cells into renal cells. Several groups have identified native renal stem cell populations, although their role in renal regeneration has not yet been well defined.

Circulating mesenchymal stem cells.

Mesenchymal precursor cells (MPCs) are multipotent cells capable of differentiating into various mesenchymal tissues, such as bone, cartilage, fat, tendon and muscle. They are present within both mesenchymal tissues and the bone marrow (BM). If marrow-derived MPCs are to have a role in repair and fibrosis of mesenchymal tissues, transit of these cells through the peripheral blood is to be expected. Although there is evidence for the existence of MPCs within the peripheral blood, results are debated and are not always reproducible. Variations in the methods of cell purification, culture and characterisation may explain the inconsistent results obtained in different studies.

A tumour that secretes glucagon-like peptide-1 and somatostatin in a patient with reactive hypoglycaemia and diabetes.

Glucagon-like peptide 1 (GLP-1), an insulinotropic hormone normally synthesised in the intestinal mucosa and released in response to a meal, is essential for normal glucose homoeostasis. There is much interest in the use of GLP-1 to treat diabetes, since the risk of hypoglycaemia is thought to be low. We report an instance of a 45-year-old woman with a GLP-1 and somatostatin secreting neuroendocrine tumour who presented with reactive hypoglycaemia and hyperglycaemia, but who was subsequently cured by surgery. This case, of a neuroendocrine tumour secreting GLP-1 and causing reactive hypoglycaemia, indicates a potential adverse effect of GLP-1 therapy for diabetes.

Synchronous roentgenographically occult lung carcinoma in patients with resectable primary lung cancer.

OBJECTIVE: To assess the prevalence of synchronous roentgenographically occult lung carcinoma (ROLC) in patients with resectable roentgenographically visible lung cancer (RVLC). METHODS: Patients undergoing surgery for RVLC in the same University Hospital were prospectively evaluated before surgery by fluorescence bronchoscopy under local anesthesia to detect synchronous ROLC. All abnormal areas, with the exception of the RVLC, had biopsies made. RESULTS: From June 1996 to January 1999, 43 patients (male/female ratio: 1.7/1.0) were evaluated before lobectomy (n = 34) or pneumonectomy (n = 10) for 44 primary RVLC. There were 10 T1N0, 19 T2N0, 1 T1N1, 9 T2N1, 1 T3N0, 3 T1N2, and 1 T3N1 lesions. The histologic type was mainly squamous carcinoma (n = 21) and adenocarcinoma (n = 14). All but two patients were smokers or ex-smokers (mean +/- SD, 48 +/- 28 pack-years). A total of 177 endobronchial biopsies were performed (4.1 +/- 2.5); 8 were too small to be informative, 43 showed non-preneoplastic alterations, and 50 were normal. There were 7 basal cell hyperplasias, 56 metaplasias, 9 dysplasias, and 4 carcinomas in situ (CIS). All the dysplasias and CIS lesions were observed in eight subjects. The synchronous CIS were treated by surgery (n = 1) or localized therapeutic modalities (n = 3). CONCLUSIONS: The high prevalence of synchronous early lung cancers (9.3%) as well as metaplasia and dysplasia in this series of patients with resectable RVLC suggests that fluorescence bronchoscopy may be a useful adjunct in the preoperative evaluation of lung cancer.

Development of a calcifying fibrous pseudotumour within a lesion of Castleman disease, hyaline-vascular subtype.

A nine year old boy with localised Castleman disease of the hyaline-vascular subtype developed a calcifying fibrous pseudotumour. This pathological association does not appear to have been described before. In this case, the development of this very unusual soft tissue tumour-like process was thought to be related to a previous fine needle aspiration biopsy, which was performed because of lymphadenopathy localised to the right inguinal area. This case provides further evidence of the reactive nature of calcifying fibrous pseudotumour and also broadens the pathological spectrum of the stromal cell proliferation that occasionally supervenes within lesions of Castleman disease, hyaline-vascular type.

Carbonic anhydrase IX antigen differentiates between preneoplastic malignant lesions in non-small cell lung carcinoma.

The MaTu interval (MN)/carbonic anhydrase (CA) IX tumour-associated antigen is a protein that is normally expressed in the gut and belongs to the carbonic anhydrase enzyme family (CA IX). It has been detected in tumour cell lines and in some solid tumours including cervical, oesophageal and clear cell renal carcinoma. This study determined MN/CA IX expression in 65 primary non-small cell lung cancer resected with curative intent and in 38 bronchial preneoplastic lesions, carcinoma in situ or microinvasive carcinoma as well as in normal bronchial tissue. The presence of MN/CA IX was detected using immunohistochemistry and Western blot analysis, whenever frozen material was available. Immunostaining was positive in 52/65 (80%) of the tumour samples. The staining was more often focal than diffuse. The percentage of stained cells in positive tumours was highly variable, ranging 1-85%. The pattern of immunostaining was predominantly cytoplasmic with a membranous reinforcement (87%). The intensity was mainly strong (69%). The presence of the protein in the tumour was confirmed by Western blot analysis in the eight samples tested. All the morphologically normal epithelia, except in close vicinity of tumours in some cases, as well as the preneoplastic bronchial lesions (basal cell hyperplasia, metaplasia and dysplasia) were immunonegative for MN/CA IX expression. In contrast, carcinoma in situ and microinvasive epithelioma showed the presence of MN-immunopositive tumoural cells in 5/7 and 4/5 of the samples, respectively. These data suggest that MN/CA IX is a useful marker for the differentiation between preneoplastic lesions and bronchial non-small cell lung cancer in the lung.

Detection of bronchial preneoplastic lesions and early lung cancer with fluorescence bronchoscopy: a study about its ambulatory feasibility under local anaesthesis.

BACKGROUND: Autofluorescence bronchoscopy (AB) enhances the bronchoscopist's ability to diagnose bronchial preneoplastic lesions and early cancer. We undertook a study to assess its feasibility and performance under local anaesthesia on a real ambulatory mode. METHODS: Thirty-four consecutive patients at very high risk for lung cancer were prospectively studied by AB under local anaesthesia, without any sedation. Lidocaine doses, time, oxygen saturation, peak expiratory flow (PEF) and the number of cough episodes were measured. Continuous assessment of the respiratory sensation was obtained with a visual analog scale. A total of 172 biopsies were performed in abnormal and normal areas. RESULTS: The procedure was long-lasting (mean +/- SD: 26.6 +/- 6.0 min), required high total doses of Lidocaine (660 +/- 107 mg) without any significant side effect, and was associated with significant decreases in O2 saturation from 98.5 +/- 1.4 to 96.1 +/- 2.5% and in PEF from 380 +/- 96 to 310 +/- 78 l/min. However, the cough counts were moderate and the majority of patients reported no respiratory discomfort. 62 hyperplasia, metaplasia, dysplasia and carcinoma in situ (CIS) were detected and the relative sensitivity of AB +/- white-light bronchoscopy (WLB) versus WLB alone was 3.75 for intraepithelial lesions corresponding to moderate dysplasia or worse. CONCLUSIONS: AB, a procedure that increases our ability to recognize preneoplastic lesions and early lung cancer, can be performed under local anaesthesia, without systemic sedation in patients at very high risk for lung cancer.

Trisomy 21 as the sole abnormality in a refractory anemia with ring sideroblasts.

Numerous chromosome abnormalities have been described in myelodysplastic syndromes, but single karyotypic aberrations are much less frequent. We report the case of a 65-year-old woman who presented a trisomy 21 as the sole karyotypic anomaly for a refractory anemia with ring sideroblasts. The nature of such an anomaly is discussed in regard to pathogenesis and prognosis.

Natural killer-like T-cell lymphoma of the stomach.

We report the case of a 69-year-old white woman who developed a natural killer (NK)-like T-cell lymphoma involving primarily the stomach. The tumour consisted of large and pleomorphic lymphocytes infiltrating the gastric mucosa. Immunohistochemistry performed on paraffin sections showed the neoplastic cells to be CD3+, CD5-, CD8-, CD43+, CD45RO+, and CD57+. In addition, these cells also expressed HLA-DR, granzyme B, and, to a lesser extent, the CD30 activation marker. No pathologic features suggesting Helicobacter pylori, Epstein-Barr virus infection, or lymphocytic gastritis were found within adjacent normal mucosa. The patient had no previous history of coeliac disease, and her serology for H. pylori was negative. Since lymphomas are usually considered the neoplastic counterpart of normal lymphocytic subsets, it is possible that in this case the tumour cells originate from a distinct cytotoxic T-cell population normally present within the gastric mucosa. The pathogenesis of this highly unusual neoplasm, however, remains a mystery.

Subcutaneous panniculitis-like T-cell lymphoma: further evidence for a distinct neoplasm originating from large granular lymphocytes of T/NK phenotype.

We report the case of a 20 year-old caucasian woman who presented a primary subcutaneous panniculitis-like T-cell lymphoma (SPTCL) as an invasive tumor of the chest wall. Herein, the neoplastic cells were found to express a CD3+CD8+ phenotype but also displayed variably the natural killer (NK)-associated antigens CD56 and CD57 as well as granzyme B. On cytological examination, these cells showed a large granular lymphocyte (LGL)-like morphology with presence of azurophilic granules in their cytoplasm. Electron dense and membrane bound granules like those found in cytotoxic T lymphocytes (CTL) were also demonstrated by electron microscopy. Neither rearrangement of the T-cell receptor subunits nor Epstein-Barr virus (EBV) genome was observed at the molecular level. The LGL-like features of the neoplastic cells found in this case and the presence of NK-associated antigens provide additional support to the cytotoxic derivation of most SPTCL.