Immunolabelling of glomerular deposits in kidney biopsies routinely fixed in glutaraldehyde

1989 ◽  
Vol 47 ◽  
pp. 910-911
Author(s):  
Ś Lhoták ◽  
I. Alexopoulou ◽  
G. T. Simon
Keyword(s):  
Uv Light ◽  
Kidney Diseases ◽  
Light Chains ◽  
Microscopical Level ◽  
Accurate Identification ◽  
Light Microscopical Level ◽  
Dense Deposits ◽  
Cacodylate Buffer ◽  
Glomerular Deposits ◽  
Prognostic Importance

Various kidney diseases are characterized by the presence of dense deposits in the glomeruli. The type(s) of immunoglobulins (Igs) present in the dense deposits are characteristic of the disease. The accurate Identification of the deposits is therefore of utmost diagnostic and prognostic importance. Immunofluorescence (IF) used routinely at the light microscopical level is unable to detect and characterize small deposits found in early stages of glomerulonephritis. Although conventional TEM is able to localize such deposits, it is not capable of determining their nature. It was therefore attempted to immunolabel at EM level IgG, IgA IgM, C3, fibrinogen and kappa and lambda Ig light chains commonly found in glomerular deposits on routinely fixed ( 2% glutaraldehyde (GA) in 0.1M cacodylate buffer) kidney biopsies.The unosmicated tissue was embedded in LR White resin polymerized by UV light at -10°C. A postembedding immunogold technique was employed

scholarly journals Quantitative Immunoprecipitation Free Light Chain Mass Spectrometry (QIP-FLC-MS) Simplifies Monoclonal Protein Assessment and Provides Added Clinical Value in Systemic AL Amyloidosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4375-4375 ◽  
Author(s):  
Faye Amelia Sharpley ◽  
Hannah Victoria Giles ◽  
Richa Manwani ◽  
Shameem Mahmood ◽  
Sajitha Sachchithanantham ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Light Chain ◽  
Residual Disease ◽  
Renal Involvement ◽  
Light Chains ◽  
Al Amyloidosis ◽  
Free Light Chains ◽  
Accurate Identification ◽  
Monoclonal Protein ◽  
Serum And Urine

Introduction Early diagnosis, effective therapy and precise monitoring are central for improving clinical outcomes in systemic light chain (AL) amyloidosis. Diagnosis and disease response assessment is primarily based on the presence of monoclonal immunoglobulins and free light chains (FLC). The ideal goal of therapy associated with best outcomes is a complete responses (CR), defined by the absence of serological clonal markers. In both instances, detection of the monoclonal component (M-component) is based on serum FLC assessment together with traditional serum and urine electrophoretic approaches, which present inherent limitations and lack sensitivity particularly in AL where the levels are typically low. Novel mass spectrometry methods provide sensitive, accurate identification of the M-component and may prove instrumental in the timely management of patients with low-level amyloidogenic light chain production. Here we assess the performance of quantitative immunoprecipitation FLC mass spectrometry (QIP-FLC-MS) at diagnosis and during monitoring of AL amyloidosis patients treated with bortezomib-based regimens. Methods We included 46 serial patients with systemic AL amyloidosis diagnosed and treated at the UK National Amyloidosis Centre (UK-NAC). All patients had detailed baseline assessments of organ function and serum FLC measurements. Baseline, +6- and +12-month serum samples were retrospectively analysed by QIP-FLC-MS. Briefly, magnetic microparticles were covalently coated with modified polyclonal sheep antibodies monospecific for free kappa light chains (anti-free κ) and free lambda light chains (anti-free λ). The microparticles were incubated with patient sera, washed and treated with acetic acid (5% v/v) containing TCEP (20 mM) in order to elute FLC in monomeric form. Mass spectra were acquired on a MALDI-TOF-MS system (Bruker, GmbH). Results were compared to serum FLC measurements (Freelite®, The Binding Site Group Ltd), as well as electrophoretic assessment of serum and urine proteins (SPE, sIFE, UPE and uIFE). Results Cardiac (37(80%) patients) and renal (31(67%) patients) involvement were most common; 25(54%) patients presented with both. Other organs involved included liver (n=12), soft tissue (n=4), gastrointestinal tract (n=3) and peripheral nervous system (n=2). Baseline Freelite, SPE, sIFE and uIFE measurements identified a monoclonal protein in 42(91%), 22(48%), 34(74%) and 21(46%) patients, respectively. A panel consisting of Freelite + sIFE identified the M-component in 100% of the samples. QIP-FLC-MS alone also identified an M-component in 100% of the samples and was 100% concordant with Freelite for typing the monoclonal FLC (8 kappa, 34 lambda). In 4 patients, QIP-FLC-MS identified an additional M-protein that was not detected by the other techniques. In addition, 4/8(50%) kappa and 4/38(11%) lambda patients showed a glycosylation pattern of monoclonal FLCs at baseline by mass spectrometry. Interestingly, the frequency of renal involvement was significantly lower for patients with non-glycosylated forms (25% vs 76%, p=0.01), while no similar relationship was found for any other organs. During the 1-year follow-up period, 17 patients achieved a CR; QIP-FLC-MS identified serum residual disease in 13(76%) of these patients. Conclusion In our series, QIP-FLC-MS was concordant with current serum methods for identifying the amyloidogenic light chain type and provided, against all other individual tests, improved sensitivity for the detection of the monoclonal protein at diagnosis and during monitoring. The ability to measure the unique molecular mass of each monoclonal protein offers clone-specific tracking over time. Glycosylation of free light chains is over-represented in AL patients which may allow earlier diagnosis and better risk-assessment of organ involvement. Persistence of QIP-FLC-MS positive M component in patients otherwise in CR may allow targeted therapy. Overall, QIP-FLC-MS demonstrates potential to be exploited as a single serum test for precise serial assessment of monoclonal proteins in patients with AL amyloidosis. Disclosures Wechalekar: GSK: Honoraria; Janssen-Cilag: Honoraria; Amgen: Research Funding; Takeda: Honoraria; Celgene: Honoraria.

Some ultrastructural aspects of Crithidia guilhermei n.sp. isolated from Phaenicia cuprina (Diptera: Calliphoridae)

1986 ◽  
Vol 64 (12) ◽  
pp. 2837-2842 ◽  
Author(s):  
Maurilio J. Soares ◽  
Reginaldo P. Brazil ◽  
Amilcar Tanuri ◽  
Wanderley de Souza
Keyword(s):  
Freeze Fracture ◽  
Kinetoplast Dna ◽  
Microscopical Level ◽  
Thin Sections ◽  
Crithidia Luciliae ◽  
Transmission Electron ◽  
Light Microscopical Level ◽  
Attachment Region ◽  
Phaenicia Sericata ◽  
A New Species

A flagellate trypanosomatid was isolated from the fly Phaenicia cuprina captured in Rio de Janeiro, Brazil. It grows well in liver infusion – trypticase medium, in the form of choanomastigotes, typical of the genus Crithidia. Morphometrical data obtained at the light microscopical level indicated that the new isolated Crithidia is smaller than Crithidia luciliae, a parasite isolated from Phaenicia sericata. Transmission electron microscopy of thin sections revealed that this trypanosomatid has a flagellar pocket divided into two compartments, one basal and the other apical, separated by a region of attachment of the flagellum to the cell body. The attachment region was characterized in freeze-fracture replicas. The flagellate has a compact kinetoplast DNA network. As in endosymbiote-containing trypanosomatids previously described, no subpellicular microtubules were seen in the regions where the mitochondria touched the plasma membrane, although no endosymbiotes were found in this flagellate. Electrophoretic mobility of six enzymes showed that the parasite could not be grouped in any of the isoenzymic pattern groups of other Crithidia spp. These observations indicate that the trypanosomatid isolated from P. cuprina is a new species of Crithidia. The flagellate is described as Crithidia guilhermei n.sp.

scholarly journals Evaluation of the Classification Accuracy of the Kidney Biopsy Direct Immunofluorescence through Convolutional Neural Networks

2020 ◽  
Vol 15 (10) ◽  
pp. 1445-1454 ◽  
Author(s):  
Giulia Ligabue ◽  
Federico Pollastri ◽  
Francesco Fontana ◽  
Marco Leonelli ◽  
Luciana Furci ◽  
...  
Keyword(s):  
Neural Networks ◽  
Convolutional Neural Networks ◽  
Kidney Biopsy ◽  
Ground Truth ◽  
Light Chains ◽  
Direct Immunofluorescence ◽  
Support Tool ◽  
Glomerular Deposits ◽  
Web Platform

Background and objectivesImmunohistopathology is an essential technique in the diagnostic workflow of a kidney biopsy. Deep learning is an effective tool in the elaboration of medical imaging. We wanted to evaluate the role of a convolutional neural network as a support tool for kidney immunofluorescence reporting.Design, setting, participants, & measurementsHigh-magnification (×400) immunofluorescence images of kidney biopsies performed from the year 2001 to 2018 were collected. The report, adopted at the Division of Nephrology of the AOU Policlinico di Modena, describes the specimen in terms of “appearance,” “distribution,” “location,” and “intensity” of the glomerular deposits identified with fluorescent antibodies against IgG, IgA, IgM, C1q and C3 complement fractions, fibrinogen, and κ- and λ-light chains. The report was used as ground truth for the training of the convolutional neural networks.ResultsIn total, 12,259 immunofluorescence images of 2542 subjects undergoing kidney biopsy were collected. The test set analysis showed accuracy values between 0.79 (“irregular capillary wall” feature) and 0.94 (“fine granular” feature). The agreement test of the results obtained by the convolutional neural networks with respect to the ground truth showed similar values to three pathologists of our center. Convolutional neural networks were 117 times faster than human evaluators in analyzing 180 test images. A web platform, where it is possible to upload digitized images of immunofluorescence specimens, is available to evaluate the potential of our approach.ConclusionsThe data showed that the accuracy of convolutional neural networks is comparable with that of pathologists experienced in the field.

scholarly journals Lowicryl K4M embedding of brain tissue for immunogold electron microscopy.

1985 ◽  
Vol 33 (9) ◽  
pp. 969-973 ◽  
Author(s):  
K L Valentino ◽  
D A Crumrine ◽  
L F Reichardt
Keyword(s):  
Electron Microscopy ◽  
Brain Tissue ◽  
Room Temperature ◽  
Uv Light ◽  
Uranyl Acetate ◽  
Immunogold Electron Microscopy ◽  
Cacodylate Buffer ◽  
Perfusion Fixation ◽  
Embedding Medium ◽  
Lowicryl K4m

We present methods for embedding brain tissue in Lowicryl K4M embedding medium and localizing antigens using postembedding immunogold techniques. After perfusion fixation with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer, blocks of rat brain were placed in 2% aqueous uranyl acetate for 1 hour, dehydrated in 50%, 70%, and 95% ethanol, infiltrated with Lowicryl/ethanol mixtures (1:2 for 10 min, 1:1 for 15 min) and 100% Lowicryl (20 min and 25 min). Polymerization was carried out under UV light for 24-48 hours at room temperature. Several neural antigens, including three different synaptic vesicle proteins and an enzyme associated with the postsynaptic density, were localized by this technique, indicating that this procedure may have wide applicability.

Precocity of the endothelial proliferation during a course of rapid goitrogenesis

1984 ◽  
Vol 105 (4) ◽  
pp. 487-491 ◽  
Author(s):  
M.-C. Many ◽  
J.-F. Denef ◽  
S. Haumont
Keyword(s):  
Endothelial Cells ◽  
Cellular Proliferation ◽  
Pulse Labelling ◽  
Follicular Cells ◽  
Microscopical Level ◽  
Light Microscopical Level ◽  
Endothelial Proliferation ◽  
C3h Mice ◽  
Thyroid Hyperplasia

Abstract. Thyroid hyperplasia was induced in C3H mice by a low iodine diet feeding supplemented with propylthiouracil. The morphological modifications associated to the development of hyperplasia were analyzed at light microscopical level and the cellular proliferation was studied by autoradiography after a pulse labelling with [3H]thymidine. The initial modification during the course of hyperplasia is the development of the vascularization. It includes the dilatation of the capillaries, which occurs before any extended modification of the follicular cells and any change of the thyroid weight, and the proliferation of endothelial cells which starts earlier than that of follicular cells.

Immunoelectron characterisation of the inter-endothelial junctions of human term placenta

1993 ◽  
Vol 104 (4) ◽  
pp. 1073-1081 ◽  
Author(s):  
L. Leach ◽  
P. Clark ◽  
M.G. Lampugnani ◽  
A.G. Arroyo ◽  
E. Dejana ◽  
...  
Keyword(s):  
Microvascular Endothelium ◽  
Microscopical Level ◽  
Human Term Placenta ◽  
Light Microscopical Level ◽  
Endothelial Junctions ◽  
Ultrathin Frozen Sections ◽  
Alpha V Beta 3 ◽  
Placental Microvessels ◽  
Fibre Matrix ◽  
Diffuse Distribution

The molecular constituents of the paracellular clefts in human placental microvessels were investigated using antibodies against PECAM-1, pan-cadherin, A-CAM (N-cadherin), cadherin-5 and two types of integrins (those recognised by antibodies to the beta 1 chain and alpha v beta 3). Ultrastructural localisation of these molecules in ultrathin frozen sections of human term placentae was attempted using colloidal gold immunocytochemistry, after establishing their presence by indirect immunofluorescence. At the light microscopical level, the endothelial paracellular clefts were found to be immunoreactive to the antibodies against PECAM-1, cadherin-5 and pan-cadherin, but not the integrins. The latter showed diffuse distribution in the endothelium and in the abluminal interstitial space. PECAM-1 and pan-cadherin were also seen in the cytoplasm and luminal surface of the endothelium. Immunoelectron studies revealed that the cadherins and PECAM-1 were present in the wide regions of the paracellular clefts, but not in tight junctional regions. Using immunocytochemistry, these wide junctional areas were found to be associated with the cytoskeletal linking molecules vinculin and alpha-actinin. These regions may therefore contain adherens-type junctions. Cadherin-5, localised by two different monoclonal antibodies, 7B4 and TEA, was the only antigen which was cleft-specific, the others also being seen in the cytoplasm of the microvascular endothelium. Cadherin-5 and pan-cadherin were co-localised in the same wide junction, but were usually seen to occupy different microdomains of, and different wide zones of, the same cleft. The cell adhesion molecules localised in the paracellular wide junctions of the human placental microvessels may play a role in maintaining the intercellular spacing between endothelial cells, and may be part of a paracellular “fibre matrix” with permeability-restricting properties.

scholarly journals Application of geostatistical functions and deep features to kidney biopsy images to differentiate focal segmental glomerulosclerosis from minimal change disease

2020 ◽  
Author(s):  
Justino Duarte Santos ◽  
Romuere R. V. Silva ◽  
Rodrigo M. S. Veras
Keyword(s):  
Focal Segmental Glomerulosclerosis ◽  
Minimal Change Disease ◽  
Kidney Diseases ◽  
Image Features ◽  
Information Criteria ◽  
Minimal Change ◽  
Kappa Index ◽  
Accurate Identification ◽  
Chronic Kidney Diseases ◽  
Segmental Glomerulosclerosis

Chronic kidney diseases arise from acute or intermittent pathologies that have not been adequately treated, such as minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). The accurate identification of these two diseases is of paramount importance, because their treatments and prognoses are different. Thus, we propose a method that is capable of differentiating MCD from FSGS based on images from pathological examinations. In the proposed method, we use four pre-trained convolutional neural networks and geostatistical functions to extract image features. Of the 8,720 extracted features, we selected 94 based on mutual information criteria, and in the classification step, we used a random forest classifier. The proposed method obtained an accuracy of 94.3% and Kappa index of 87.9%, a level that is regarded as “almost perfect”, confirming that our method is very promising.

scholarly journals Oligosecretory monoclonal gammopathy with renal involvement

2016 ◽  
Vol 88 (12) ◽  
pp. 82-87
Author(s):  
L V Kozlovskaya ◽  
V V Rameev ◽  
T V Androsova ◽  
I N Kogarko ◽  
B S Kogarko ◽  
...  
Keyword(s):  
Monoclonal Gammopathy ◽  
Kidney Diseases ◽  
Renal Involvement ◽  
Diagnostic Techniques ◽  
Light Chains ◽  
Free Light Chains ◽  
Detection Rates ◽  
Highly Sensitive ◽  
Separate Form ◽  
Monoclonal Proteins

The article deals with the so-called monoclonal gammopathy of undetermined significance (MGUS), which is being actively explored in the world and has been recently investigated in Russia. It indicates the principles of identifying the phenotypes of MGUS and criteria for assessing the risk of its progression to cancer. There is an update on the possible involvement of monoclonal proteins in the pathogenesis of certain non-neoplastic kidney diseases, renal injuries in particular. The paper gives their classification and enumerates differential diagnostic techniques, including the Freelite method, a highly sensitive one to determine free light chains (FLC), prognostic criteria, and approaches to treating each separate form in relation to the phenotype of a monoclonal protein. The authors present their own data on detection rates for MGUS at a multidisciplinary hospital and a clinical case of MGUS-associated membranoproliferative glomerulonephritis, by justifying a treatment regimen containing bortezomib (velcade).

Seminal Phagocytes Detection at Light-Microscopy Level using Three Different Techniques

1996 ◽  
Vol 54 ◽  
pp. 778-779
Author(s):  
L. A. Benítez ◽  
O. G. Díaz ◽  
R. M. M. Eguía ◽  
M. G. A. Gallegos
Keyword(s):  
Male Infertility ◽  
Light Microscopy ◽  
Reproductive Tract ◽  
Neutral Red ◽  
Microscopical Level ◽  
Light Microscopical Level ◽  
Semithin Sections ◽  
Inflammatory Processes ◽  
Infertile Patients

The phagocytes are normally present in the conective tissue at different organs, and their number increased in immunological or inflammatory processes. In these cases, they are present too in corporal fluids.The presence of phagocytes in semen was referred by several authors, but that phenomenon has not been suficiently analysed. At smear Papanicolaou stained analysis, the seminal phagocytes can be inadverted thus, their relationship to the male infertility or another reproductive tract pathology can not be established.In this study we implemented three techniques to detect the presence of phagocytes in semen of 28 infertile patients: Myeloperoxidase for polymorphonuclears (leukocyte), Neutral red for macrophages and the semithin sections analysis at light microscopical level of the seminal pellet obtained by centrifugation and included in epoxyresin.

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