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Background: When immunofluorescence on the frozen section is insufficient or unavailable, salvage immunofluorescence techniques can be used on formalin-fixed, paraffin-embedded tissue. The goal of the current investigation was to evaluate the diagnostic value of paraffin immunofluorescence following proteinase K digestion on skin biopsy samples in comparison to fresh frozen immunofluorescence. Materials and Methods: It was standardized and compared to the immunofluorescence on fresh frozen tissue (IF-Frozen) for paraffin immunofluorescence by proteinase K digestion of formalin-fixed paraffin-embedded skin biopsies (IF-FFPE). The study included 50 native skin biopsy cases, and fluorescein isothiocyanate-labeled IgA, IgG, IgM, and C3 intensity levels were evaluated in each case. Results: A total of 50 cases of native skin biopsy were included in the study, and their intensities for IgA, IgG, IgM, and C3 antibodies were compared. The average staining intensities in each disease group for the antibodies had equal intensity or had a minor difference (1+)/significant difference (2+). Paraffin immunofluorescence, proteinase K digestion had the best correlation, that is, had either equal or minor difference (1+) with fresh frozen immunofluorescence. The difference of 2+ intensity of antibodies between IF-FFPE and IF-Frozen was noted mainly in C3 antibody on paraneoplastic pemphigus. IF-FFPE showed a sensitivity of 100%, 97.6%, 100%, and 81.6% for IgA, IgG, IgM, and C3, respectively, whereas the specificity was 100% for IgA, IgG, IgM, and C3. Limitations: Small sample size and and the employment of one method of antigen retrieval in IF-FFPE. Conclusion: Immunofluorescence techniques done on formalin-fixed paraffin-embedded tissue can serve as salvage techniques in cases where immunofluorescence on the frozen section may not be adequate or may not be available.
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Background Immunofluorescence techniques done on formalin-fixed, paraffin-embedded tissue can serve as salvage techniques in cases where immunofluorescence on the frozen section may not be adequate or available. The present study was undertaken to assess the diagnostic utility of paraffin immunofluorescence by proteinase K digestion on renal biopsy compared to fresh frozen immunofluorescence. Methodology The paraffin immunofluorescence by proteinase K digestion of paraffin-embedded renal biopsy (IF-FFPE) was standardized and compared with the immunofluorescence on fresh frozen tissue (IF-Frozen). A total of 50 cases of the native renal biopsy were included in the study, and their intensity for fluorescein isothiocyanate-labeled IgA, IgG, IgM, C3, kappa, and lambda was compared. Results A total of 50 cases of the native renal biopsy were included in the study, and their intensity for fluorescein isothiocyanate-labeled antibodies of IgA, IgG, IgM, C3, kappa, and lambda was compared. The difference of 2+ intensity of antibodies between IF-FFPE and IF-Frozen was noted mainly in lupus nephritis (15%), followed by IgA nephropathy (10%) and membranoproliferative glomerulonephritis (7%). IF-FFPE showed a sensitivity of 90.3%, 91.8%, 82.7%, 81.1%, 92.1%, and 94.6% for IgA, IgG, IgM, C3, kappa, and lambda, respectively, whereas specificity was 100% for IgA, IgG, C3, kappa, and lambda and 95.2% for IgM. Conclusions Immunofluorescence techniques done on formalin-fixed, paraffin-embedded tissue can serve as salvage techniques in kidney biopsies.
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There is a growing interest in expanding the multiplexing capability of immunohistochemistry to achieve a deeper phenotyping of various cell types in health and disease. Here, we describe a protocol of cyclic multiplex fluorescent immunohistochemistry that enables the labeling of up to 16 antigens on the same formalin-fixed paraffin-embedded section using "off-the-shelf," commercially available, primary antibodies as well as fluorescently conjugated secondary antibodies. Key steps include the denaturing/stripping of the antibodies by microwaving and the quenching of any remaining fluorescent signal between the cycles of otherwise traditional multiplexed fluorescent immunohistochemistry. We have successfully applied this protocol to characterize astrocytic and microglial responses to Aß plaques and neurofibrillary tangles in Alzheimer's disease brains, but it could be easily adapted to other user's needs regarding cell types, disease, and organ.
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Enfermedad de Alzheimer , Humanos , Inmunohistoquímica , Adhesión en Parafina , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Neuroglía/metabolismo , Formaldehído/metabolismo , FenotipoRESUMEN
In 1979, the author in his younger days experienced an autobiographical case of idiopathic rhabdomyolysis. The heme casts in formalin-fixed, paraffin-embedded sections of the kidney were immunoreactive for myoglobin. In these days, the immunoperoxidase technique had been utilized as a research seed by using paraformaldehyde-fixed frozen sections. The precious experience prompted the young author of his younger days to apply the immunoperoxidase method to diagnostic pathology using formalin-fixed paraffin-embedded sections. A brief history of early development of chromogenic immunostaining in diagnostic pathology in Japan is summarized.
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Objective: To observe the effect of immunofluorescence double staining for foamy macrophages and Mycobacterium tuberculosis (MTB) in paraffin-embedded tissue of clinical tuberculous wound, in comparison with three routine staining methods. Methods: The experimental method was used. From April 2019 to May 2020, 10 patients with tuberculous wound (5 males and 5 females, aged 28-77 years) meeting the inclusion criteria were treated in the Department of Burns and Plastic & Wound Repair Surgery of Xiang'an Hospital of Xiamen University. The paraffin-embedded wound tissue were collected during extended debridement and preserved in the Department of Pathology of this hospital. Forty paraffin sections were made from the wound tissue of each patient. Hematoxylin-eosin (HE) staining, immunohistochemical staining, Ziehl-Neelsen and immunohistochemical double staining, immunofluorescence double staining were performed respectively, with 10 sections in each method. The section rejection rate of four staining methods were calculated. The recognition and detection of wound granuloma tissue in the four staining methods were observed and counted, and the recognition and detection of foamy macrophages in the wound tissue stained with four methods were observed. The MTB detection in the wound granuloma tissue and non-granuloma tissue in the four staining methods were compared. The subtyping and distribution of foamy macrophages and detection rate of MTB in the wound granuloma tissue and non-granuloma tissue, the morphologic clarity of foamy macrophages, as well as the non-specific staining rate and the loss rate of positive reaction of MTB and foamy macrophages by Ziehl-Neelsen and immunohistochemical double staining were compared with those of immunofluorescence double staining. Data were statistically analyzed with Fisher's exact probability test, one-way analysis of variance, independent sample t test and Wilcoxon signed rank test. Results: The section rejection rate of HE staining, immunohistochemical staining, Ziehl-Neelsen and immunohistochemical double staining, and immunofluorescence double staining were 3% (3/100), 1% (1/100), 6% (6/100), and 2% (2/100), respectively. There was no statistically significant difference among the four groups (P=0.26). All the four staining methods could identify granuloma tissue, and the number of granuloma structures was similar (F=1.284, P=0.28). All the four staining methods were able to identify foamy macrophages in the wound tissue, which was detected in each section. No MTB was observed in the wound granuloma tissue or non-granuloma tissue by HE staining or immunohistochemical staining. MTB was observed distributing in the wound granuloma tissue and non-granuloma tissue by Ziehl-Neelsen and immunohistochemical double staining and immunofluorescence double staining, and most MTB distributed in the wound granuloma tissue. Ziehl-Neelsen and immunohistochemical double staining could not distinguish foamy macrophages engulfed MTB from that non-engulfed MTB. Immunofluorescence double staining showed that foamy macrophages engulfed MTB mostly distributed in the wound granuloma tissue, and the foamy macrophages non-engulfed MTB mostly distributed in the wound non-granuloma tissue. The detection rates of MTB in wound granuloma and non-granuloma tissue in immunofluorescence double staining were (89.00±0.08)% and (82.67±0.05)%, respectively, which were significantly higher than (54.56±0.14)% and (44.44±0.13)% in Ziehl-Neelsen and immunohistochemical double staining (t=-12.495, -7.961, P<0.01). Compared with that of Ziehl-Neelsen and immunohistochemical double staining, immunofluorescence double staining showed better foamy macrophages clarity in wound tissue (Z=-3.162, P<0.01). The nonspecific staining rate and positive reaction loss rate of MTB and foamy macrophages in wound tissue of immunofluorescence double staining were (9.11±0.07)% and (9.22±0.07)%, respectively, which were significantly lower than (20.67±0.06)% and (44.00±0.12)% of Ziehl-Neelsen and immunohistochemical double staining (t=4.569, 15.519, P<0.01). Conclusions: Compared with HE staining, immunohistochemical staining, and Ziehl-Neelsen and immunohistochemical double staining, the immunofluorescence double staining is easy to operate, giving clear and intuitive images. It allows accurate imaging co-localization of MTB and foamy macrophages in paraffin-embedded tissue of clinical tuberculous wound.
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Mycobacterium tuberculosis , Adulto , Anciano , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Macrófagos , Masculino , Persona de Mediana Edad , Adhesión en Parafina , Coloración y EtiquetadoRESUMEN
BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is a rare dermal mesenchymal tumor known as a low-grade, slow-growing malignancy. The local invasion and high rate of recurrence following surgical treatment are the main concerns to plan the best surgical approach of treatment. AIMS: In the current study, it is aimed to provide an experience of slow-Mohs surgery for the treatment of patients with DSFP. PATIENTS/METHODS: Number of 25 patients with the diagnosis of DFSP through histological and immunostaining study was included. The slow-Mohs was performed by excision of the tumor with margins accounting for 1-2 cm from both the tumor margins and three-dimensional thickness. The obtained tissue margins were horizontally, and if any of the specimens was not margin-free, the procedure was repeated. The patients' opinion about the procedure was assessed using Patient-Observer Scar Assessment Scale (POSAS). RESULTS: Number of 25 patients were included and followed for a median of 46.9 months. The median of the area of excision was 35.56 cm2 , and the median clinical excision margins were 19 mm (tumor excision margins + thickness of the three-dimensional excision). The surgery was performed once for 16 (64%), and postoperative skin closure within 5-7 days after the procedure was performed for 19 (76%) patients. None of the patients represented any recurrence. The patients' overall opinion and satisfaction POSAS score accounted for 2.3 ± 1.65 and 1.6 ± 0.59, respectively. CONCLUSION: The findings of the current study are in favor of slow-Mohs surgery for the management of DFSP, while more extensive studies are strongly recommended for generalization of this procedure.
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Dermatofibrosarcoma , Neoplasias Cutáneas , Estudios de Cohortes , Dermatofibrosarcoma/cirugía , Estudios de Seguimiento , Humanos , Cirugía de Mohs , Recurrencia Local de Neoplasia/cirugía , Estudios Retrospectivos , Neoplasias Cutáneas/cirugíaRESUMEN
BACKGROUND: Dermatofibrosarcoma protuberans is a rare malignant tumor with a high rate of recurrence after surgery. Moh's micrographic surgery allows examination of all surgical margins to ensure complete removal. OBJECTIVE: To evaluate the use of Moh's micrographic surgery using paraffin-embedded sections for the treatment of dermatofibrosarcoma protuberans. METHODS: We performed a retrospective analysis of 33 patients with dermatofibrosarcoma protuberans treated in our department with paraffin-embedded micrographic surgery between January 2002 and June 2015. Our cases included patients with primary untreated disease and also those with persistent disease previously treated surgically elsewhere, with histologically positive margins. RESULTS: Tumors were most commonly located on the trunk. After the first stage of micrographic surgery, including an initial lateral margin, 20 (60.6%) tumors were completely excised, 11 (33.3%) tumors required two stages and one tumor each (3.0%) required 4 and 6 stages respectively. Patients were monitored for recurrence for a mean duration of 6.5 years. There was no recurrence in any of our 33 patients. CONCLUSIONS: Our results indicate that Moh's micrographic surgery with paraffin-embedded sections may be the method of choice to treat dermatofibrosarcoma protuberans with a low recurrence rate, while preserving surrounding normal healthy tissue.
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Dermatofibrosarcoma/diagnóstico , Dermatofibrosarcoma/cirugía , Cirugía de Mohs/métodos , Adhesión en Parafina/métodos , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Germinal centers are short-lived microanatomical compartments with essential roles in adaptive immunity. These lymphoid structures can be identified in secondary lymphoid organs using both flow cytometry and immunohistological analyses, but only the latter provides useful architectural and spatial information. Here we describe how to use immunofluorescence and immunohistochemistry with specific antibodies to precisely highlight the cellular and architectural features of germinal centers, both in human and mouse secondary lymphoid organs, and to study their normal development and disturbance in disease.
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Técnica del Anticuerpo Fluorescente , Centro Germinal/citología , Centro Germinal/inmunología , Inmunohistoquímica , Inmunidad Adaptativa , Animales , Antígenos/inmunología , Biomarcadores , Técnica del Anticuerpo Fluorescente/métodos , Centro Germinal/metabolismo , Inmunohistoquímica/métodos , RatonesRESUMEN
Immunohistochemistry is a major technique to determine the distribution and localization of differentially produced proteins in the context of an intact tissue. It exploits one of the properties of antibodies, specific binding to an antigen, i.e., to the epitope of its target protein, in combination with a color-developing enzymatic reaction or tagged fluorophore. We have clarified the spatial and temporal expression patterns of CCN family proteins in several different types of animal tissues by using this immunohistochemical technique to support our corresponding data obtained in vitro. In this chapter, we provide our protocol for immunohistochemistry optimized for paraffin-embedded sections after having determined the optimal conditions for the use of antibodies against each member of the CCN family.
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Proteínas CCN de Señalización Intercelular/metabolismo , Inmunohistoquímica , Animales , Proteínas CCN de Señalización Intercelular/genética , Expresión Génica , Inmunohistoquímica/métodos , RatonesRESUMEN
During a long time, immunofluorescence has been neglected to benefit of molecular biology especially genetics, transcriptomics, and proteomics analyses. These techniques give good results on cell culture but for organs that are made of numerous cells with several compartments, various states of differentiation as in epidermis, immunohistochemistry is always relevant. Double (triple) staining by immunofluorescence allows positive cells identification in complex cell structure (for example, pericytes and endothelial cells in vessels) and subcellular localizations. In order to, due to improvement of antibodies avoiding especially species cross-reactions, microscopy and specific softwares, quality of staining, and acquired images have been upgraded. Consequently, this technique permits, as molecular biology analyses, quantification of the level of expression as intensity of fluorescence can be measured in each cells and each compartments (nuclear, cytoplasmic). In order to immunofluorescence on cells and tissue needs few materials and gives at the same times qualitative and quantitative results and must be used more widely especially when a mutation was associated to a disease.
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Proteínas CCN de Señalización Intercelular/metabolismo , Células Epidérmicas , Epidermis/metabolismo , Técnica del Anticuerpo Fluorescente , Piel/citología , Piel/metabolismo , Proteínas CCN de Señalización Intercelular/genética , Humanos , Inmunohistoquímica/métodos , Queratinocitos/metabolismo , Melanocitos/metabolismoRESUMEN
The renal microvasculature is targeted during aging, sometimes producing chronic kidney disease (CKD). Overdiagnosis of CKD in older persons is concerning. To prevent it, a new concept of "healthy aging" is arising from a healthy renal donor study. We investigated the renal microcirculatory changes of three older persons and compared them with that of one patient with nephrosclerosis using a three-dimensional (3D) reconstruction technique that we previously developed. This method uses a virtual slide system and paraffin-embedded serial sections of surgical material that was double-immunostained by anti-CD34 and anti-α smooth muscle actin (SMA) antibodies for detecting endothelial cells and medial smooth muscle cells, respectively. In all cases, the 3D images proved that arteriosclerotic changes in large proximal interlobular arteries did not directly induce distal arterial change or glomerulosclerosis. The nephrosclerotic patient showed severe hyalinosis with luminal narrowing of small arteries directly inducing glomerulosclerosis. We also visualized an atubular glomerulus and intraglomerular dilatation of an afferent arteriole during healthy aging on the 3D image and showed that microcirculatory changes were responsible for them. Thus, we successfully visualized healthy aged kidneys on 3D images and confirmed the underlying pathology. This method has the ability to investigate renal microcirculatory damage during healthy aging.
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Adenocarcinoma de Células Claras/diagnóstico por imagen , Envejecimiento/patología , Carcinoma de Células Renales/diagnóstico por imagen , Imagenología Tridimensional/métodos , Glomérulos Renales/diagnóstico por imagen , Neoplasias Renales/diagnóstico por imagen , Microvasos/diagnóstico por imagen , Nefroesclerosis/diagnóstico por imagen , Actinas/genética , Actinas/metabolismo , Adenocarcinoma de Células Claras/irrigación sanguínea , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma de Células Claras/ultraestructura , Anciano , Anciano de 80 o más Años , Envejecimiento/metabolismo , Antígenos CD34/genética , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/ultraestructura , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Expresión Génica , Humanos , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/ultraestructura , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/metabolismo , Neoplasias Renales/ultraestructura , Masculino , Microtomía , Microvasos/metabolismo , Microvasos/ultraestructura , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/ultraestructura , Nefroesclerosis/metabolismo , Nefroesclerosis/patología , Adhesión del TejidoRESUMEN
We have developed a new virtual microscopy method, with two- and three-dimensional (2D, 3D) synchronization, that enables visualization of the human renal microvasculature. The method was used to evaluate 120-150 serially cut sections of paraffin-embedded human renal tissue from nephrectomized samples. Virtual microscopy images of sections double-immunostained with antibodies against CD34 (an endothelium marker) and smooth muscle actin (an arterial media marker) and stained with periodic acid-Schiff were processed using digital imaging analysis software. Image registration was conducted to generate 3D displays with red-green-blue color segmentation. The reconstructed images of the microvasculature, including the interlobular arteries and the glomeruli, allowed visualization of 3D structures and direct glomerular connections. Synchronizing these 3D images with the corresponding 2D images revealed the relationships between arteriosclerotic lesions and downstream glomeruli. Thus, interlobular arteries with moderate intimal thickening and afferent arterioles with segmental hyalinosis/sclerosis, as seen on the 2D images, exhibited wall irregularities on the corresponding 3D images. However, these lesions were not directly influenced by lesions in downstream glomeruli, such as sclerotic lesions. Our virtual-slide method based on 2D and 3D image synchronization provides a comprehensive view of the renal microcirculation and therefore novel insights into the pathogenesis of vascular-associated renal diseases.
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Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Glomérulos Renales/patología , Microcirculación/fisiología , Microvasos/patología , Insuficiencia Renal Crónica/patología , Humanos , Imagenología Tridimensional/métodos , Riñón/patología , Masculino , Insuficiencia Renal Crónica/diagnóstico , Túnica Media/patologíaRESUMEN
BACKGROUND: Apart from infection with human filariae, zoonotic filariasis also occurs worldwide, and the numbers of cases have been increasing steadily. Diagnosis of intact filariae in tissues or organs depends on histological identification. The morphology of parasites in tissue-embedded sections is poor and shows high levels of homoplasy. Thus, the use of morphological characteristics in taxonomic studies is difficult and may not allow a specific diagnosis. METHODS: Here we report the use of real-time PCR with high-resolution melting analysis (HRM) to detect and identify Brugia malayi, Brugia pahangi, Wuchereria bancrofti, and Dirofilaria immitis in paraffin-embedded sections. Assay specificity was determined using other tissue-dwelling parasites, Angiostrongylus cantonensis, Gnathostoma spinigerum, and Cysticercus cellulosae. We also developed a quick paraffin removal protocol. RESULTS: Both human and animal filariae in formalin-fixed paraffin-embedded sections (FFPES) were diagnosed and identified rapidly, whereas other parasites were negative. There was no difference in the melting temperature of products amplified from filarial DNA obtained from unstained FFPES and Hematoxylin & Eosin-stained sections. Therefore, the DNA extraction protocols developed in this study could be used for real-time PCR with HRM. CONCLUSIONS: We report the successful application of a HRM-PCR assay to differentiate four filarial parasites in FFPES, thus providing the pathologist with an effective alternative diagnostic procedure. Furthermore, the quick paraffin removal protocol developed could shorten the duration and number of steps required for paraffin removal using a standard protocol.
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Brugia Malayi/aislamiento & purificación , Brugia pahangi/aislamiento & purificación , Dirofilaria immitis/aislamiento & purificación , Filariasis/parasitología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Wuchereria bancrofti/aislamiento & purificación , Animales , Brugia Malayi/genética , Brugia pahangi/genética , ADN de Helmintos/aislamiento & purificación , Dirofilaria immitis/genética , Femenino , Filariasis/patología , Humanos , Adhesión en Parafina , Sensibilidad y Especificidad , Wuchereria bancrofti/genética , ZoonosisRESUMEN
INTRODUCTION: Dermatofibrosarcoma protuberans (DFSP) is a potentially malignant dermal mesenchymal tumour with a high risk of local recurrence. DFSP presents a sprawling appearance whose complete excision requires important margins. DFSP was initially resected with a 5cm excision margins, and more recently 3cm then 2cm margins were recommended. Mohs micrographic surgery (MMS) helps reduce these margins thanks to a 3-dimensional excision around the tumour, which is analysed in its entirety. We used the modified MMS called slow-MMS and tried every time it was possible to perform direct closure. METHODS: Thirty-five patients presenting a DFSP between 2004 and 2013 within the Plastic Surgery unit at Claudius Regaud Institute were included in this retrospective study. The patients were treated with slow-MMS using paraffin-embedded sections. RESULTS: One surgery was necessary for 72% of patients. For 17%, we had to perform a second surgery, and for 11% a third one. Our median clinical excision margins was 17mm (range 9.0:30.0). After a median follow-up of 46 months (range 35.2:60.2), we didn't observe any recurrence. Only one case required a local flap; for the others, the loss of substance was resolved with a direct closure. CONCLUSION: Slow-MMS enabled a local control of the margins without recurrence at 46 months in our series. Besides, it helps performing smaller margins than wide excision and thus preserving the tissues. In our opinion, this is the treatment of choice regarding DFSP for which tissue sparing is essential. It seems particularly appropriate near functional areas or on the face.