Suitability of Fourier transform infrared microscopy for the diagnosis of cystic echinococcosis in human tissue sections.

Cystic echinococcosis (CE) is a global health concern caused by cestodes, posing diagnostic challenges due to nonspecific symptoms and inconclusive radiographic results. Diagnosis relies on histopathological evaluation of affected tissue, demanding comprehensive tools. In this retrospective case study, Fourier transform infrared microscopy was explored for detecting and identifying CE through biochemical changes in human tissue sections. Tissue samples from 11 confirmed CE patients were analyzed. Archived FFPE blocks were cut and stained, and then CE-positive unstained sections were examined using Fourier transform infrared microscopy post-deparaffinization. Results revealed the method's ability to distinguish echinococcus elements from human tissue, irrespective of organ type. This research showcases the potential of mid-infrared microscopy as a valuable diagnostic tool for CE, offering promise in enhancing diagnostic precision in the face of the disease's complexities.

Deep learning analysis of mid-infrared microscopic imaging data for the diagnosis and classification of human lymphomas.

The present study presents an alternative analytical workflow that combines mid-infrared (MIR) microscopic imaging and deep learning to diagnose human lymphoma and differentiate between small and large cell lymphoma. We could show that using a deep learning approach to analyze MIR hyperspectral data obtained from benign and malignant lymph node pathology results in high accuracy for correct classification, learning the distinct region of 3900 to 850 cm-1 . The accuracy is above 95% for every pair of malignant lymphoid tissue and still above 90% for the distinction between benign and malignant lymphoid tissue for binary classification. These results demonstrate that a preliminary diagnosis and subtyping of human lymphoma could be streamlined by applying a deep learning approach to analyze MIR spectroscopic data.

Immune cell dynamics deconvoluted by single-cell RNA sequencing in normothermic machine perfusion of the liver.

Normothermic machine perfusion (NMP) has emerged as an innovative organ preservation technique. Developing an understanding for the donor organ immune cell composition and its dynamic changes during NMP is essential. We aimed for a comprehensive characterization of immune cell (sub)populations, cell trafficking and cytokine release during liver NMP. Single-cell transcriptome profiling of human donor livers prior to, during NMP and after transplantation shows an abundance of CXC chemokine receptor 1+/2+ (CXCR1+/CXCR2+) neutrophils, which significantly decreased during NMP. This is paralleled by a large efflux of passenger leukocytes with neutrophil predominance in the perfusate. During NMP, neutrophils shift from a pro-inflammatory state towards an aged/chronically activated/exhausted phenotype, while anti-inflammatory/tolerogenic monocytes/macrophages are increased. We herein describe the dynamics of the immune cell repertoire, phenotypic immune cell shifts and a dominance of neutrophils during liver NMP, which potentially contribute to the inflammatory response. Our findings may serve as resource to initiate future immune-interventional studies.

Visible- and near-infrared hyperspectral imaging for the quantitative analysis of PD-L1+ cells in human lymphomas: Comparison with fluorescent multiplex immunohistochemistry.

INTRODUCTION: We analyzed the expression of PD-L1 in human lymphomas using hyperspectral imaging (HSI) compared to visual assessment (VA) and conventional digital image analysis (DIA) to strengthen further the value of HSI as a tool for the evaluation of brightfield-based immunohistochemistry (IHC). In addition, fluorescent multiplex immunohistochemistry (mIHC) was used as a second detection method to analyze the impact of a different detection method. MATERIAL AND METHODS: 18 cases (6 follicular lymphomas and 12 diffuse large B-cell lymphomas) were stained for PD-L1 by IHC and for PD-L1, CD3, and CD8 by fluorescent mIHC. The percentage of positively stained cells was evaluated with VA, HSI, and DIA for IHC and VA and DIA for mIHC. Results were compared between the different methods of detection and analysis. RESULTS: An overall high concordance was found between VA, HSI, and DIA in IHC (Cohens Kappa = 0.810VA/HSI, 0.710 VA/DIA, and 0.516 HSI/DIA) and for VAmIHCversus DIAmIHC (Cohens Kappa = 0.894). Comparing IHC and mIHC general agreement differed depending on the methods compared but reached at most a moderate agreement (Cohens Kappa between 0.250 and 0.483). This is reflected by the significantly higher percentage of PD-L1+ cells found with mIHC (pFriedman = 0.014). CONCLUSION: Our study shows a good concordance for the different analysis methods. Compared to VA and DIA, HSI proved to be a reliable tool for assessing IHC. Understanding the regulation of PD-L1 expression will further enlighten the role of PD-L1 as a biomarker. Therefore it is necessary to develop an instrument, such as HSI, which can offer a reliable and objective evaluation of PD-L1 expression.

Visible and Near-Infrared hyperspectral imaging (HSI) can reliably quantify CD3 and CD45 positive inflammatory cells in myocarditis: Pilot study on formalin-fixed paraffin-embedded specimens from myocard obtained during autopsy.

INTRODUCTION: To implement Hyperspectral Imaging (HSI) as a tool for quantifying inflammatory cells in tissue specimens by the example of myocarditis in a collective of forensic patients. MATERIAL AND METHODS: 44 consecutive patients with suspected myocardial inflammation at autopsy, diagnosed between 2013 and 2018 at the Institute of ForensicMedicine, Medical University of Innsbruck, were selected for this study. Using the IMEC SNAPSCAN camera, visible and near infrared hyperspectral images were collected from slides stained with CD3 and CD45 to assess quantity and spatial distribution of positive cells. Results were compared with visual assessment (VA) and conventional digital image analysis (DIA). RESULTS: Finally, specimens of 40 patients were evaluated, of whom 36 patients (90%) suffered from myocarditis, two patients (5%) had suspected healing/healed myocarditis, and two did no have myocarditis (5%). The amount of CD3 and CD45 positive cells did not differ significantly between VA, HSI, and DIA (pVA/HSI/DIA = 0.46 for CD3 and 0.81 for CD45). Cohens Kappa showed a very high correlation between VA versus HSI, VA versus DIA, and HSI versus DIA for CD3 (Cohens Kappa = 0.91, 1.00, and 0.91, respectively). For CD45 an almost as high correlation was seen for VA versus HSI and HSI versus DIA (Cohens Kappa = 0.75 and 0.70) and VA versus DIA (Cohens Kappa = 0.89). CONCLUSION: HSI is a reliable and objective method to count inflammatory cells in tissue slides of suspected myocarditis. Implementation of HSI in digital pathology might further expand the possibility of a sophisticated method.

The future of pathology is digital.

Information, archives, and intelligent artificial systems are part of everyday life in modern medicine. They already support medical staff by mapping their workflows with shared availability of cases' referral information, as needed for example, by the pathologist, and this support will be increased in the future even more. In radiology, established standards define information models, data transmission mechanisms, and workflows. Other disciplines, such as pathology, cardiology, and radiation therapy, now define further demands in addition to these established standards. Pathology may have the highest technical demands on the systems, with very complex workflows, and the digitization of slides generating enormous amounts of data up to Gigabytes per biopsy. This requires enormous amounts of data to be generated per biopsy, up to the gigabyte range. Digital pathology allows a change from classical histopathological diagnosis with microscopes and glass slides to virtual microscopy on the computer, with multiple tools using artificial intelligence and machine learning to support pathologists in their future work.

Borrelia and Chlamydia Can Form Mixed Biofilms in Infected Human Skin Tissues.

Our research group has recently shown that Borrelia burgdorferi, the Lyme disease bacterium, is capable of forming biofilms in Borrelia-infected human skin lesions called Borrelia lymphocytoma (BL). Biofilm structures often contain multiple organisms in a symbiotic relationship, with the goal of providing shelter from environmental stressors such as antimicrobial agents. Because multiple co-infections are common in Lyme disease, the main questions of this study were whether BL tissues contained other pathogenic species and/or whether there is any co-existence with Borrelia biofilms. Recent reports suggested Chlamydia-like organisms in ticks and Borrelia-infected human skin tissues; therefore, Chlamydia-specific polymerase chain reaction (PCR) analyses were performed in Borrelia-positive BL tissues. Analyses of the sequence of the positive PCR bands revealed that Chlamydia spp. DNAs are indeed present in these tissues, and their sequences have the best identity match to Chlamydophila pneumoniae and Chlamydia trachomatis. Fluorescent immunohistochemical and in situ hybridization methods demonstrated the presence of Chlamydia antigen and DNA in 84% of Borrelia biofilms. Confocal microscopy revealed that Chlamydia locates in the center of Borrelia biofilms, and together, they form a well-organized mixed pathogenic structure. In summary, our study is the first to show Borrelia-Chlamydia mixed biofilms in infected human skin tissues, which raises the questions of whether these human pathogens have developed a symbiotic relationship for their mutual survival.

Clinical infrared microscopic imaging: An overview.

New developments in Mid-infrared microscopic imaging instrumentation and data analysis have turned this method into a conventional technique. This imaging method offers a global analysis of samples, with a resolution close to the cellular level enabling the acquisition of local molecular expression profiles. It is possible to get chemo-morphological information about the tissue status, which represents an essential benefit for future analytical interpretation of pathological changes of tissue. In this review, we give an overview of Mid-infrared microscopic imaging and its applications in clinical research.

[Pityriasis amiantacea and folliculitis decalvans : An unusual manifestation associated with antitumor necrosis factor-α therapy]. / Pityriasis amiantacea und Folliculitis decalvans : Eine ungewöhnliche Erscheinung, assoziiert mit Anti-Tumor-Nekrose-Faktor-α-Therapie.

We report on a 21-year-old woman with a 3-year history of crusts and erosions on her scalp that had appeared after starting treatment with adalimumab due to Crohn's disease. By clinicopathological correlation pityriasis amiantacea with underlying folliculitis decalvans was diagnosed. Topical and systemic antibiotic treatment showed rapid response. The occurrence of pityriasis amiantacea in folliculitis decalvans associated with tumor necrosis factor (TNF)-α inhibitor therapy is remarkable and highlights the ambivalent role of TNF-α in diseases with immunological dysfunctions in combination with infections.

Benefits and limitations of belatacept in 4 hand-transplanted patients.

Belatacept (cytotoxic T-lymphocyte-associated protein 4 Ig) is an emerging treatment in kidney transplantation. Lack of nephrotoxicity and possibly an inhibitory effect on the development of donor-specific antibodies (DSAs) make it an interesting agent in hand transplantation. To reduce calcineurin inhibitor immunosuppression and preserve kidney function, we have added belatacept to the therapeutic regimen of 4 hand-transplanted patients at month 4 and at 6, 9, and 13 years after hand-forearm transplantation. Patients received 5 mg/kg belatacept every 2 weeks, and the dosing interval was extended to 4 weeks after 5 applications. Belatacept was initially well tolerated in all cases. Two patients were weaned to a low-dose tacrolimus monotherapy together with monthly belatacept applications. One patient is taking belatacept with lowered tacrolimus and sirolimus trough levels. A fourth patient had significant levels of DSAs at time of conversion and progressed to a severe necrotizing rejection early despite an unaltered baseline immunosuppression. Finger skin necrosis and histologic signs of severe chronic allograft vasculopathy eventually led to amputation of the graft. Implementation of belatacept can be beneficial in hand transplantation. However, our findings indicated both potential and caution and reflection of the immunologic state at the time of conversion.

Evidence of In Vivo Existence of Borrelia Biofilm in Borrelial Lymphocytomas.

Lyme borreliosis, caused by the spirochete Borrelia burgdorferi sensu lato, has grown into a major public health problem. We recently identified a novel morphological form of B. burgdorferi, called biofilm, a structure that is well known to be highly resistant to antibiotics. However, there is no evidence of the existence of Borrelia biofilm in vivo; therefore, the main goal of this study was to determine the presence of Borrelia biofilm in infected human skin tissues. Archived skin biopsy tissues from borrelial lymphocytomas (BL) were reexamined for the presence of B. burgdorferi sensu lato using Borrelia-specific immunohistochemical staining (IHC), fluorescent in situ hybridization, combined fluorescent in situ hybridization (FISH)-IHC, polymerase chain reaction (PCR), and fluorescent and atomic force microscopy methods. Our morphological and histological analyses showed that significant amounts of Borrelia-positive spirochetes and aggregates exist in the BL tissues. Analyzing structures positive for Borrelia showed that aggregates, but not spirochetes, expressed biofilm markers such as protective layers of different mucopolysaccharides, especially alginate. Atomic force microscopy revealed additional hallmark biofilm features of the Borrelia/alginate-positive aggregates such as inside channels and surface protrusions. In summary, this is the first study that demonstrates the presence of Borrelia biofilm in human infected skin tissues.

[Actinic Keratosis]. / Aktinische Keratose.

Actinic keratosis is a cutaneous lesion characterized by proliferation of atypical epidermal keratinocytes due to prolonged exposure to exogenous factors such as ultraviolet radiation. AKs are in-situ-squamous cell carcinomas (PEC) of the skin. AK typically presents as erythematous, scaly patch or papule (classic AK), occasionally as thick, adherent scale on an erythematous base. Mostly fair-skinned adults are affected. AKs typically occur in areas of frequent sun exposure (balding scalp, face, "H-region", lateral neck, décolleté, dorsum of the hand and lower extremities). Actinic Cheilitis is the term used for AKs appearing on the lips. The diagnosis of AK is based on clinical examination including inspection and palpation. The typical palpable rough surface of AK often precedes a visible lesion. Dermoscopy may provide additional information. If diagnosis is uncertain and invasion suspected, biopsy and histopathologic evaluation should be performed. The potential for progression to invasive PECs mandates therapeutic intervention. Treatment options include topical and systemic therapies. Topical therapies are classified into physical, medical and combined physical-chemical approaches and a sequential combination of treatment modalities is possible. Topical-physical cryotherapy is the treatment of choice for isolated, non-hypertrophic AK. Topical-medical treatment, e. g. 5-fluoruracil (5FU) cream or Imiquomod or Ingenolmebutat application is used for multiple, non-hypertrophic AKs. For hypertrophic AKs, a dehorning pretreatment with salicinated vaseline is recommended. Isolated hypertrophic AKs often need cryotherapy with prolonged freezing time or several consecutive applications. Sequentially combined approaches are recommended for multiple, hypertrophic AKs. Photodynamic therapy (PDT) as example for a combined physical-chemical approach is an established treatment for multiple, non-hypertrophic and hypertrophic AKs. Prevention includes avoidance of sun and application of sunscreen with high sun protection factor. Regular clinical checkups aid in early recognition of AKs.

Rapamycin and CTLA4Ig synergize to induce stable mixed chimerism without the need for CD40 blockade.

The mixed chimerism approach achieves donor-specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti-CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor-specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC-mismatched/minor antigen-matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non-MHC antigens cause graft rejection and split tolerance.

[Clinical presentation of cutaneous adnexal tumors]. / Klinik der Hautadnextumoren.

Cutaneous adnexal lesions can sometimes be clinically diagnosed even by an experienced clinician or a differential diagnosis can at least be narrowed down. However, clinical findings alone cannot replace histological investigations and diagnosis or make them superfluous. This expertise is based on an algorithm which first differentiates inflammatory pseudo-tumors, such as ruptured infundibular cysts (atheroma) from authentic neoplastic adnexal lesions. In a second step criteria of regularity and/or chaos, such as asymmetry, irregular border, color variation and/or destruction with exulceration help to evaluate the dignity. In a third step criteria of differentiation allow the characterization of lesions varying in size from macules to papules, plaques, nodules and tumors to the subgroups of adnexal differentiation. Infundibular differentiation is characterized by comedones and is skin-colored, yellow or white and hard. Follicular differentiation notifies hair and is skin-colored, pearl-like to occasionally brown-black and variably hard. Sebaceous differentiation signifies lobulation and is yellow to skin-colored or red and soft. Apocrine lesions are reddish and fleshy. Eccrine differentiation shows either papillary reddish-brown (differential diagnosis viral warts) or skin-colored hard lesions. Multiple, monomorphous lesions are characteristic of syndromes, such as Spiegler-Brooke-Fend, Birt-Hogg-Dubé, Muir-Torre, and Gorlin-Goltz.One peculiarity of adnexal lesions is their potential to form cysts. Cysts with horny or hairy material are skin-colored to yellow, with glandular fluid fluctuation, a bluish character, and with illumination a Tyndall phenomenon becomes obvious, while ruptured cysts reveal an erythematous-reddish, ill-defined foreign body reaction. Brown to bluish-gray and black color is seen by the presence of melanocytes with melanin in lesions with mostly follicular differentiation. Strong vascularization and bleeding are reddish, soft, spongy and compressible and in due course variably dark due to the presence of hemosiderin.

Panniculitides, an algorithmic approach.

The issue of inflammatory diseases of subcutis and its mimicries is generally considered a difficult field of dermatopathology. Yet, in my experience, with appropriate biopsies and good clinicopathological correlation, a specific diagnosis of panniculitides can usually be made. Thereby, knowledge about some basic anatomic and pathological issues is essential. Anatomy differentiates within the panniculus between the fatty lobules separated by fibrous septa. Pathologically, inflammation of panniculus is defined and recognized by an inflammatory process which leads to tissue damage and necrosis. Several types of fat necrosis are observed: xanthomatized macrophages in lipophagic necrosis; granular fat necrosis and fat micropseudocysts in liquefactive fat necrosis; mummified adipocytes in "hyalinizing" fat necrosis with/without saponification and/or calcification; and lipomembranous membranes in membranous fat necrosis. In an algorithmic approach the recognition of an inflammatory process recognized by features as elaborated above is best followed in three steps: recognition of pattern, second of subpattern, and finally of presence and composition of inflammatory cells. Pattern differentiates a mostly septal or mostly lobular distribution at scanning magnification. In the subpattern category one looks for the presence or absence of vasculitis, and, if this is the case, the size and the nature of the involved blood vessel: arterioles and small arteries or veins; capillaries or postcapillary venules. The third step will be to identify the nature of the cells present in the inflammatory infiltrate and, finally, to look for additional histopathologic features that allow for a specific final diagnosis in the language of clinical dermatology of disease involving the subcutaneous fat.

Fourier transform infrared imaging analysis in discrimination studies of squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) of the oral cavity and oropharynx represents more than 95% of all malignant neoplasms in the oral cavity. Histomorphological evaluation of this cancer type is invasive and remains a time consuming and subjective technique. Therefore, novel approaches for histological recognition are necessary to identify malignancy at an early stage. Fourier transform infrared (FTIR) imaging has become an essential tool for the detection and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of tumor progression. FTIR imaging is a modern analytical technique enabling molecular imaging of a complex biological sample and is based on the absorption of IR radiation by vibrational transitions in covalent bonds. One major advantage of this technique is the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification, and separation steps. With this imaging technique, it is possible to obtain unique images of the spatial distribution of proteins, lipids, carbohydrates, cholesterols, nucleic acids, phospholipids, and small molecules with high spatial resolution. Analysis and visualization of FTIR imaging datasets are challenging and the use of chemometric tools is crucial in order to take advantage of the full measurement. Therefore, methodologies for this task based on the novel developed algorithm for multivariate image analysis (MIA) are often necessary. In the present study, FTIR imaging and data analysis methods were combined to optimize the tissue measurement mode after deparaffinization and subsequent data evaluation (univariate analysis and MIAs). We demonstrate that it is possible to collect excellent IR spectra from formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMAs) of OSCC tissue sections employing an optimised analytical protocol. The correlation of FTIR imaging to the morphological tissue features obtained by histological staining of the sections demonstrated that many histomorphological tissue patterns can be visualized in the colour images. The different algorithms used for MIAs of FTIR imaging data dramatically increased the information content of the IR images from squamous cell tissue sections. These findings indicate that intra-operative and surgical specimens of squamous cell carcinoma tissue can be characterized by FTIR imaging.

[Focal nodular hyperplasia in a 12-year-old boy: case report and considerations]. / Fokale noduläre Hyperplasie bei einem 12-jährigen Jungen: Fallbericht und Themenbetrachtung.

BACKGROUND: Focal nodular hyperplasia (FNH) is a benign hepatic tumor of unknown origin. It is only observed rarely in children (approximately 1-2% of all pediatric liver tumors). CASE REPORT: A 12-year-old boy who suffered from infectious mononucleosis with liver involvement and hepatomegaly underwent a sonographic scan of the liver at an external hospital 3 months after the infection disappeared which revealed a tumor of the left hepatic lobule. Subsequent further examination (abdominal CT and MRT scans) confirmed the diagnosis of a highly vascularized mass about 10 cm in diameter, suspicious for FNH. Due to the high vascularization no biopsy was performed. A preoperative angiographic coiling and complete surgical resection was carried out because of the size and morphologic uncertainty. The diagnosis of FNH was confirmed by histological examination. The annual sonographic examination at follow-up has been uneventful for a 4-year period. CONCLUSIONS: Due to the rarity the diagnosis of FNH in children can be difficult leading to differential diagnostic problems. Due to the risk of bleeding in larger size tumors a biopsy is a point of controversy. Complete resection and histopathological examination of FNHs in childhood is a mandatory therapeutic option, which may be indicated in large tumors or, as in the present case tumors of uncertain biological behaviour.

Comparative analysis of immunohistochemistry, polymerase chain reaction and focus-floating microscopy for the detection of Treponema pallidum in mucocutaneous lesions of primary, secondary and tertiary syphilis.

BACKGROUND: The incidence of syphilis is increasing in many parts of the world including a re-emergence in Western Europe and North America. Depending on the disease stage, direct detection of Treponema pallidum in mucocutaneous lesions of syphilis may be difficult and histopathological findings are not always straightforward. Thus, the correct histological diagnosis may be challenging. OBJECTIVES: Comparatively to evaluate the evidence for infection with T. pallidum by immunohistochemistry (IHC), polymerase chain reaction (PCR) and focus-floating microscopy (FFM). METHODS: A series of 86 paraffin-embedded skin biopsy samples from patients with primary, secondary or tertiary syphilis was assessed for detection of T. pallidum by IHC and FFM; 45 specimens were also investigated by a T. pallidum-specific PCR analysis. Histopathological reaction patterns and number and distribution of treponemes were studied, and all data were re-evaluated by clinicopathological correlation. RESULTS: Using a polyclonal antibody directed against T. pallidum, we detected the presence of T. pallidum by IHC in 42/86 (49%) samples [6/9 (67%) primary, 34/62 (55%) secondary and 2/15 (13%) tertiary syphilis]. T. pallidum-specific DNA was detected in 31/45 (69%) specimens [4/4 (100%) primary, 26/34 (76%) secondary and 1/7 (14%) tertiary syphilis]. In comparison, FFM analysis resulted in an overall detection rate of 82/86 (95%) [9/9 (100%) primary, 60/62 (97%) secondary and 13/15 (87%) tertiary syphilis]. Significant differences were observed concerning amount and distribution of organisms (epitheliotropic vs. endotheliotropic) in correlation to the three disease stages and to histopathological reaction patterns. CONCLUSIONS: FFM is a highly sensitive and specific method to detect T. pallidum in tissue from mucocutaneous syphilis lesions. Our results indicate that a combination of PCR and FFM, as the most sensitive approach, could provide an additional benefit for the histopathological diagnosis of (late) secondary and tertiary syphilis and may be helpful in cases where serological testing of T. pallidum antibodies has failed, but the clinical suspicion for syphilis remains.