Role of the mechanisms for antibody repertoire diversification in monoclonal light chain deposition disorders: when a friend becomes foe.

The adaptive immune system of jawed vertebrates generates a highly diverse repertoire of antibodies to meet the antigenic challenges of a constantly evolving biological ecosystem. Most of the diversity is generated by two mechanisms: V(D)J gene recombination and somatic hypermutation (SHM). SHM introduces changes in the variable domain of antibodies, mostly in the regions that form the paratope, yielding antibodies with higher antigen binding affinity. However, antigen recognition is only possible if the antibody folds into a stable functional conformation. Therefore, a key force determining the survival of B cell clones undergoing somatic hypermutation is the ability of the mutated heavy and light chains to efficiently fold and assemble into a functional antibody. The antibody is the structural context where the selection of the somatic mutations occurs, and where both the heavy and light chains benefit from protective mechanisms that counteract the potentially deleterious impact of the changes. However, in patients with monoclonal gammopathies, the proliferating plasma cell clone may overproduce the light chain, which is then secreted into the bloodstream. This places the light chain out of the protective context provided by the quaternary structure of the antibody, increasing the risk of misfolding and aggregation due to destabilizing somatic mutations. Light chain-derived (AL) amyloidosis, light chain deposition disease (LCDD), Fanconi syndrome, and myeloma (cast) nephropathy are a diverse group of diseases derived from the pathologic aggregation of light chains, in which somatic mutations are recognized to play a role. In this review, we address the mechanisms by which somatic mutations promote the misfolding and pathological aggregation of the light chains, with an emphasis on AL amyloidosis. We also analyze the contribution of the variable domain (VL) gene segments and somatic mutations on light chain cytotoxicity, organ tropism, and structure of the AL fibrils. Finally, we analyze the most recent advances in the development of computational algorithms to predict the role of somatic mutations in the cardiotoxicity of amyloidogenic light chains and discuss the challenges and perspectives that this approach faces.

AL(light chain)-amyloidogenesis by mesangial cells involves active participation of lysosomes: An ultrastructural study.

Amyloid formation by cells is a stepwise process that occurs in macrophages and cells capable of transforming into a macrophage phenotype. One such cell is the mesangial cell in the kidney. It has been shown that mesangial cells are engaged in AL (light chain associated)- amyloidogenesis after transforming phenotypically from a smooth muscle to a macrophage phenotype. The actual process of amyloid fibril formation has not been dissected. This ultrastructural study which includes the examination of lysosomal gradient specimens addresses this issue by analyzing the sequence of events that takes place as fibrils are formed in endosomes and lysosomes. The findings indicate that fibrillogenesis begins in endosomes but is completed and most pronounced in the lysosomal compartment. As early as 10 min after incubation of human mesangial cells with AL-LCs, amyloid fibrils are formed in endosomes but mostly occurs in the mature lysosomal compartment. This is the first time that fibril formation is demonstrated experimentally occurring inside human mesangial cells and the entire sequence of events taking place is elucidated.

Scalp Metastasis as an Initial Manifestation of Squamous Cell Carcinoma of the Lung: Case Report of an Extremely Rare Entity.

Cutaneous metastasis of primary visceral neoplasm is an unusual phenomenon. However, cutaneous metastasis as an initial presentation of clinically silent visceral neoplasm is exceedingly rare. We are reporting a unique case of an elderly male patient who presented with a solitary scalp metastasis as an initial manifestation of underlying lung cancer. Further diagnostic evaluation revealed neoplastic primary lung disease. This case report emphasizes the importance of physicians being aware of these unusual clinical presentations of visceral malignancies. It is also critical to order appropriate diagnostic tests promptly to establish an accurate diagnosis and begin the proper treatment for a better prognosis. Skin lesions can be a diagnostic manifestation of lung cancer and predict a poor prognosis. We conclude that in patients with a history of smoking or lung cancer who present with cutaneous lesions, the possibility of skin metastasis of primary lung cancer should always be considered in the differential diagnosis.

Glomerulopathic Light Chain-Mesangial Cell Interactions: Sortilin-Related Receptor (SORL1) and Signaling.

INTRODUCTION: Deciphering the intricacies of the interactions of glomerulopathic Ig light chains with mesangial cells is key to delineate signaling events responsible for the mesangial pathologic alterations that ensue. METHODS: Human mesangial cells, caveolin 1 (CAV1), wild type (WT) ,and knockout (KO), were incubated with glomerulopathic light chains purified from the urine of patients with light chain-associated (AL) amyloidosis or light chain deposition disease. Associated signaling events induced by surface interactions of glomerulopathic light chains with caveolins and other membrane proteins, as well as the effect of epigallocatechin-3-gallate (EGCG) on the capacity of mesangial cells to intracellularly process AL light chains were investigated using a variety of techniques, including chemical crosslinking with mass spectroscopy, immunofluorescence, and ultrastructural immunolabeling. RESULTS: Crosslinking experiments provide evidence suggesting that sortilin-related receptor (SORL1), a transmembrane sorting receptor that regulates cellular trafficking of proteins, is a component of the receptor on mesangial cells for glomerulopathic light chains. Colocalization of glomerulopathic light chains with SORL1 in caveolae and also in lysosomes when light chain internalization occurred, was documented using double immunofluorescence and immunogold labeling ultrastructural techniques. It was found that EGCG directly blocks c-Fos cytoplasmic to nuclei signal translocation after interactions of AL light chains with mesangial cells, resulting in a decrease in amyloid formation. CONCLUSION: Our findings document for the first time a role for SORL1 linked to glomerular pathology and signaling events that take place when certain monoclonal light chains interact with mesangial cells. This finding may lead to novel therapies for treating renal injury caused by glomerulopathic light chains.

Understanding Mesangial Pathobiology in AL-Amyloidosis and Monoclonal Ig Light Chain Deposition Disease.

Patients with plasma cell dyscrasias produce free abnormal monoclonal Ig light chains that circulate in the blood stream. Some of them, termed glomerulopathic light chains, interact with the mesangial cells and trigger, in a manner dependent of their structural and physicochemical properties, a sequence of pathological events that results in either light chain-derived (AL) amyloidosis (AL-Am) or light chain deposition disease (LCDD). The mesangial cells play a key role in the pathogenesis of both diseases. The interaction with the pathogenic light chain elicits specific cellular processes, which include apoptosis, phenotype transformation, and secretion of extracellular matrix components and metalloproteinases. Monoclonal light chains associated with AL-Am but not those producing LCDD are avidly endocytosed by mesangial cells and delivered to the mature lysosomal compartment where amyloid fibrils are formed. Light chains from patients with LCDD exert their pathogenic signaling effect at the cell surface of mesangial cells. These events are generic mesangial responses to a variety of adverse stimuli, and they are similar to those characterizing other more frequent glomerulopathies responsible for many cases of end-stage renal disease. The pathophysiologic events that have been elucidated allow to propose future therapeutic approaches aimed at preventing, stopping, ameliorating, or reversing the adverse effects resulting from the interactions between glomerulopathic light chains and mesangium.

Tubular Injury and Dendritic Cell Activation Are Integral Components of Light Chain-Associated Acute Tubulointerstitial Nephritis.

CONTEXT.­: Light chain-associated acute tubulointerstitial nephritis (LC-ATIN) is a variant of light chain proximal tubulopathy (LCPT). It is characterized by interstitial inflammation with tubulitis and deposition of monoclonal light chains in the tubulointerstitium. LC-ATIN is a rather poorly recognized pattern of LCPT and not much is known about this entity. OBJECTIVE.­: To determine the clinicopathologic features of patients with LC-ATIN and investigate the proximal tubular injury and mechanism of interstitial inflammation in LC-ATIN. DESIGN.­: A total of 38 cases of LC-ATIN were identified from the archives of 5043 renal biopsy specimens. In all cases, routine light microscopic examination, immunofluorescence, and electron microscopic examination were performed. In selected cases, immunofluorescent staining of dendritic cells and immunohistochemical staining for 4 tubular injury markers-KIM-1, p53, bcl-2, and Ki-67-were performed. RESULTS.­: A characteristic finding in LC-ATIN cases was immunofluorescence staining of monoclonal light chains along tubular basement membranes in linear fashion and inside proximal tubular cells with a granular pattern. No monoclonal light chains were present in glomerular or vascular compartments confirmed with immunofluorescence, electron microscopy, and ultrastructural gold labeling. Ten of 15 LC-ATIN cases (67%) were concurrently positive for the 4 tubular injury markers. Dendritic cells were identified within the tubulointerstitium in the renal biopsy specimens, interacting with surrounding tubules with light-chain deposits and inflammatory cells. CONCLUSIONS.­: Significant proximal tubular injury occurs associated with LC-ATIN, and the monoclonal light chains accumulated in proximal tubular cells contribute to the injury. Dendritic cells are involved in the pathogenesis of interstitial inflammation in LC-ATIN.

Cystathione ß-Synthase Is Increased in Thyroid Malignancies.

BACKGROUND: Cystathione ß-synthase (CBS) catalyzes the conversion of homocysteine and cysteine to hydrogen sulfide (H2S) and cystathione, via the trans-sulfuration pathway. CBS protein expression levels are increased in several different human malignancies, with increased protein expression correlating with parameters such as tumor stage, anaplasia, metastases, and chemotherapy resistance. MATERIALS AND METHODS: This study employed tissue microarrays to examine CBS expression in benign thyroid tissue, thyroid oncocytomas, thyroid follicular adenomas, and in follicular, papillary, anaplastic, and medullary thyroid carcinomas. RESULTS: CBS expression was increased in all thyroid carcinomas types compared to benign thyroid tissue, but not in thyroid follicular adenomas or oncocytomas. A similar pattern was observed for nicotinamide phosphoribosyltransferase (NAMPT) tissue microarray analysis comparing thyroid adenomas and follicular carcinomas. CONCLUSION: For the first time, we showed that an H2S-syntheszing enzyme plays a role in thyroid malignancies. Additionally, our data suggest that CBS and NAMPT immunohistochemistry may be useful in differentiating follicular adenomas from follicular carcinomas.

Metastatic TFE3-overexpressing renal clear cell carcinoma with dense granules: a histological, immunohistochemical, and ultrastructural study.

This is a case report of a 46-year-old white male who presented with dyspnea. Thoracic and abdominal examinations showed a heterogeneously enhancing mass in the right kidney, multiple pulmonary nodules, and left pleural thickening with large pleural effusion. Pleura biopsy revealed a malignant neoplasm composed of cells with predominantly clear cytoplasm. Considering the large mass in the right kidney, clear cell renal cell carcinoma (RCC) was the main differential diagnosis. The diagnosis in this case was not definitive by histology alone since clear cell RCC markers such as RCC and AE1/AE3 were negative, and CD10 was only focally positive. Transcription factor E3 (TFE3) immunohistochemistry was positive, while the XP11.2 translocation testing was negative. Electron microscopy demonstrated that the tumor cells had abundant cytoplasmic glycogen and lipid, focal long microvilli lining rare lumina, and adjacent interdigitating cell membranes joining the neoplastic cells, indicating a diagnosis of renal clear cell carcinoma. In addition, numerous crystalline-like dense granules were identified in the cytoplasm of the neoplastic cells, which are reminiscent of those typically seen in alveolar soft part sarcoma and rarely described in XP11.2 translocation RCC. Overall, this renal tumor likely represents a variant of XP11.2 translocation RCC, overexpressing TFE3 with dense granules.

Parasitic leiomyoma presenting as an inguinal hernia in a postmenopausal woman.

Uterine leiomyomas are one of the most common tumors affecting reproductive-age women. Leiomyomas can present as an intrauterine mass or rarely as an extrauterine tumor. Depending on its location, the diagnosis of extrauterine leiomyoma can be challenging, and multiple imaging modalities may be needed for correct identification and differentiation from malignant entities. We report the case of a 48-year-old-postmenopausal female who presented with a painful left inguinal mass, which was clinically diagnosed as inguinal hernia. Ultrasound, computed tomography, magnetic resonance imaging, and percutaneous biopsy were used to characterize the mass. Surgical resection and histopathological analysis revealed the mass to be a parasitic leiomyoma, a very rare cause of inguinal hernia, especially in a postmenopausal woman.

Malignant granular cell tumors: the role of electron microscopy in the definitive diagnosis of an extremely aggressive soft tissue neoplasm.

Granular cell tumors (GCTs) are rare soft tissue neoplasms which may be multicentric. The vast majority are benign, however approximately 100 malignant GCTs have been reported, with only 8 originating in the vulva. Malignant GCTs are very aggressive with very poor survival rates. As the diagnosis of malignant GCT carries an extremely poor prognosis, the utilization of EM ensures that the most accurate diagnosis possible can be rendered.

Phenotypic plasticity of mesenchymal stem cells is crucial for mesangial repair in a model of immunoglobulin light chain-associated mesangial damage.

Mesangiopathies produced by glomerulopathic monoclonal immunoglobulin light chains (GLCs) acting on the glomerular mesangium produce two characteristic lesions: AL-amyloidosis (AL-Am) and light chain deposition disease (LCDD). In both cases, the pathology is centered in the mesangium, where initial and progressive damage occurs. In AL-Am the mesangial matrix is destroyed and replaced by amyloid fibrils and in LCDD, the mesangial matrix is increased and remodeled. The collagen IV rich matrix is replaced by tenascin. In both conditions, mesangial cells (MCs) become apoptotic as a direct effect of the GLCs. MCs were incubated in-vitro with GLCs and animal kidneys were perfused ex-vivo via the renal artery with GLCs, producing expected lesions, and then mesenchymal stem cells (MSCs) were added to both platforms. Each of the two platforms provided unique information that when put together created a comprehensive evaluation of the processes involved. A "cocktail" with growth and differentiating factors was used to study its effect on mesangial repair. MSCs displayed remarkable phenotypic plasticity during the repair process. The first role of the MSCs after migrating to the affected areas was to dispose of the amyloid fibrils (in AL-Am), the altered mesangial matrix (in LCDD) and apoptotic MCs/debris. To accomplish this task, MSCs transformed into facultative macrophages acquiring an abundance of lysosomes and endocytotic capabilities required to engage in phagocytic functions. Once the mesangial cleaning was completed, MSCs transformed into functional MCs restoring the mesangium to normal. "Cocktail" made the repair process more efficient.

Animal models of monoclonal immunoglobulin-related renal diseases.

The renal deposition of monoclonal immunoglobulins can cause severe renal complications in patients with B cell and plasma cell lymphoproliferative disorders. The overproduction of a structurally unique immunoglobulin can contribute to the abnormal propensity of monoclonal immunoglobulins to aggregate and deposit in specific organs. A wide range of renal diseases can occur in multiple myeloma or monoclonal gammopathy of renal significance, including tubular and glomerular disorders with organized or unorganized immunoglobulin deposits. The development of reliable experimental models is challenging owing to the inherent variability of immunoglobulins and the heterogeneity of the pathologies they produce. However, although imperfect, animal models are invaluable tools to understand the molecular pathogenesis of these diseases, and advances in creating genetically modified animals might provide novel approaches to evaluate innovative therapeutic interventions. We discuss the strategies employed to reproduce human monoclonal immunoglobulin-induced kidney lesions in animal models, and we highlight their advantages and shortcomings. We also discuss how these models have affected the management of these deposition diseases and might do so in the future. Finally, we discuss hypotheses that explain some limitations of the various models, and how these models might improve our understanding of other nephropathies without immunoglobulin involvement that have similar pathogenic mechanisms.

Oral foregut duplication cysts: A rare and fascinating congenital lesion. Case report and review of the literature.

Oral foregut duplication cysts are extremely rare lesions with approximately 57 cases reported. They are congenital cysts, located in the anterior or ventral tongue, and occur predominantly in males. They are lined by one or more types of epithelia which is limited to gastric, intestinal or respiratory epithelium. The differential diagnosis includes lymphangioma, hemangioma, ranula, epidermoid cyst, teratoma and less likely a malignant process.

Healing the damaged mesangium in nodular glomerulosclerosis using mesenchymal stem cells (MSCs): Expectations and challenges.

It has been shown experimentally that mesenchymal stem cells (MSCs) can be delivered to the mesangium in some conditions such as amyloidosis to clear debris and foreign material, and eventually transform into functional mesangial cells (MCs) and change the altered mesangial areas into normal collagen IV-rich matrix. A more challenging situation is when the matrix is rich in abnormal extracellular matrix proteins, especially those difficult to destroy such as tenascin, and, as a result, assumes a nodular appearance - what is known in pathology jargon as nodular glomerulosclerosis. MSCs find it difficult to dispose of the altered mesangial constituents, an initial step required for mesangial repair to occur successfully. The ability of MSCs to repair damaged mesangium represents a novel therapeutic intervention to reverse mesangial injury and is potentially a powerful and unique approach to prevent progression ending in end-stage renal disease (ESRD). This review will highlight progress that has been made in glomerular, and more specifically mesangial, repair, and will address future expectations and challenges to be confronted as the use of MSCs continues to be explored as a potential application for clinical practice.

Animal Models of Light Chain Deposition Disease Provide a Better Understanding of Nodular Glomerulosclerosis.

BACKGROUND: Light chain deposition disease (LCDD) is a model of glomerulosclerosis. The mature lesion of LCDD mimics nodular glomerulosclerosis in diabetic nephropathy. The pathogenetic mechanisms involved are similar in both disorders, though the causative factors are entirely different. This fact highlights the generic response of the mesangium to varied stimuli. In-vitro work has provided much insight into the pathogenesis of glomerulosclerosis in LCDD where the mesangium is the main target for initiation and progression of the disease. The lack of animal models has prevented the development of further therapeutic approaches to be tested in platforms such as ex-vivo and in-vivo preparing the way for human studies. METHODS: Light chains (LCs) obtained from the urine of patients with renal biopsy proven LCDD were delivered to glomeruli using ex-vivo and in-vivo approaches to address whether in-vitro information could be validated in-vivo. Selected in-vitro studies were conducted to address specific issues dealing with mesangial cell (MC) differentiation and composition of extracellular matrix to add additional data to the existing vast literature. Using light, electron and scanning microscopy together with immunohistochemistry and ultrastructural immunolabeling, MCs incubated in Matrigel with LCDD LCs, as well as delivery of such LCs by perfusion via renal artery (ex-vivo) and penile dorsal vein (in-vivo) to the kidneys, validation of pathogenetic pathways previously suggested in in-vitro experiments were tested and confirmed. RESULTS: The animal models described in this manuscript provide validation for the in-vitro data that have been previously published and expand our appreciation of the important role that caveolin-1 plays in signaling events essential for the downstream sequence of events that eventually leads to the pathological alterations centered in the mesangium characterized by an increase in matrix production and formation of mesangial nodules. CONCLUSIONS: The same findings observed in renal biopsies of patients with LCDD (mesangial expansion with increased matrix) were documented in the ex-vivo and in-vivo platforms. In-vivo understanding of the pathogenesis of mesangial glomerulosclerosis, as accomplished in the reported research, is crucial for the design of novel therapeutic approaches to treat a number of glomerulopathies with similar pathogenetic mechanisms. Inhibiting interactions between glomerulopathic LCs and MCs or interrupting the protein production/secretion pathways are potentially effective therapeutic maneuvers. The results obtained with caveolin-1 knockout mice emphasized the importance of caveolin-1 in signaling events essential to effect downstream mesangial alterations.

Neuroendocrine tumors in the ovary: histogenesis, pathologic differentiation, and clinical presentation.

OBJECTIVE: Primary neuroendocrine tumors in the ovary are rare. These tumors arise from the neuroendocrine cell system of ovarian stroma and surface epithelium, and may also arise from teratoma. We present four primary ovarian neuroendocrine tumors and compare clinicopathologic findings based on tumor histogenesis and site of origin. DESIGN: Four primary ovarian neuroendocrine tumors were identified from our 10-year departmental archives. H&E slides and immunostains were reviewed and the diagnoses were confirmed. Clinical history, imaging studies, and follow-up data were obtained from medical records. RESULTS: Patients' ages ranged from 26 to 63. All patients presented with abdominal discomfort and unilateral or bilateral ovarian masses. MRI and CT scans from cases 1 and 2 revealed a solid ovarian mass with no extra-ovarian extension. In case 1, the patient also had a cystic mass in the opposite ovary and an elevated urine 5-HIAA. Microscopically, case 1 revealed a well-differentiated carcinoid tumor with no surface epithelial involvement, and a mature teratoma in the contralateral ovary. Case 2 revealed a stromal carcinoid within the ovarian parenchyma. Imaging studies from cases 3 and 4 showed large complex masses with peritoneal implants and ascites. In both cases 3 and 4, tumor grossly involved both ovarian parenchyma and surface epithelium with multiple pelvic implants. In addition, liver metastases were present in case 4. Microscopically, these tumors were poorly differentiated carcinoma with neuroendocrine differentiation. Histologic sections revealed extensive necrosis, and both cases showed positivity for neuroendocrine markers. CONCLUSIONS: Primary neuroendocrine tumors in the ovary are rare and consist of a group of heterogeneous malignancies that express similar immunohistochemical markers. Primary neuroendocrine tumors that are limited to the ovarian parenchyma often arise from ovarian stroma and teratoma, and are carcinoid tumors with a good prognosis. Neuroendocrine tumors that arise from surface epithelium or dedifferentiate from de novo carcinoma often involve both ovarian stroma and surface epithelium and clinically present as aggressive malignancies with poor prognoses.

Immunotactoid Glomerulopathy and Cryoglobulinemic Nephropathy: Two Entities with Many Similarities. A Unified Conceptual Approach.

Immunotactoid glomerulopathy is a rare disorder that has been characterized at the ultrastructural level. Due to its rarity, there are few comprehensive studies relating to this disorder. Electron microscopy essentially characterizes this disease. The glomerular electron dense deposits which are typical of this condition consist of aggregates of highly organized microtubular structures of various diameters, but generally measuring 30-50 nm in width with a propensity to dispose themselves in parallel bundles intersecting in different planes. This study compares a large series of patients with cryoglobulinemic nephropathy with a series of patients with immunotactoid glomerulopathy to address whether there may be similarities that warrant considering these two entities part of a spectrum. This study reviews the clinicopathologic features of both entities and emphasizes ultrastructural findings that characterize them. Significant immunomorphologic overlap was found when these two disorders were compared in this study. There were also striking similarities in clinical presentation/behavior, laboratory findings and prognosis. Proteomic analysis has also demonstrated similar spectra for both entities. We postulate that immunotactoid glomerulopathy and cryoglobulinemic nephropathy are part of the spectrum of renal manifestations in patients with circulating cryoglobulins and renal disease.

Comparison of in vivo probe-based confocal laser endomicroscopy with histopathology in lung cancer: A move toward optical biopsy.

BACKGROUND AND OBJECTIVE: The development of novel technologies has increased the yield from transbronchial biopsies while preserving patient safety by guiding biopsies to the area of interest. Other technologies have helped identify pre-cancerous or sessile lesions in the endobronchial space by utilizing interactions between tissue and light at varying wavelengths. Probe-based confocal laser endomicroscopy (pCLE) is a new technology that encompasses the benefits of both guided biopsies and novel optical imaging in one device. This project compares pCLE images to the findings of light microscopy in non-small cell lung cancer (NSCLC). METHODS: Patients who underwent bronchoscopies between July 2012 and January 2013 for evaluation of pulmonary lesions (transbronchial and endobronchial) were recruited. Histopathological images from malignant lesions were compared with the pCLE images obtained from the same area. The microscopic and pCLE images were reviewed side by side with the microscopic findings. RESULTS: Images from pCLE correlate with some histopathological findings. pCLE changes seen in NSCLC consist of mottled elastin, septal studding and disorganization/fragmentation with increased friability. These changes also seem to correlate with degrees of differentiation. CONCLUSIONS: pCLE can identify changes to the elastin composition of the airways and alveoli in lung cancer. These changes correlate with histopathology and may help indicate the presence of malignant changes in vivo.

The many faces of cryoglobulinemic nephropathy: a clinico-pathologic study of 47 cases with emphasis on the value of electron microscopy.

The clinical and pathologic manifestations of cryoglobulinemic nephropathy (CN) are heterogeneous. The role that electron microscopy plays in the diagnosis of CN has not been properly evaluated. The main objective of this study was to define the value of ultrastructural evaluation in the diagnosis of CN. Although most of the CN cases in this series exhibited glomerular pathology with a membranoproliferative pattern, a significant number of the cases showed less well-defined morphologic patterns on light microscopic examination (i.e. mesangial proliferation, hyaline thrombi in glomerular capillaries without significant cellular proliferation, exudative glomerulonephritis, etc). Immunofluorescence microscopy also revealed variable immunoglobulin and complement component patterns, some with "full-house" expression of immunoreactants. A subset of these CN cases was associated with light chain restriction. Thus, differential diagnosis can be a challenge as many other glomerulonephritis overlap in immunomorphologic characteristics. Because the immunomorphologic manifestations of CN are so varied, confirmation of a suspected diagnosis of CN or making a diagnosis in a less than a typical immunomorphologic setting required careful ultrastructural evaluation to find unequivocal diagnostic findings or at least supportive evidence in the form of detection of substructure in the electron dense glomerular deposits consistent with cryoglobulins. Even in cases where the light and immunofluorescence findings in suspicious clinical situations were quite consistent with CN, electron microscopy provided the final proof to substantiate the diagnosis in the great majority of the cases.