Mitotic Patterns Research Articles

Specific Mitotic Events Drive Cytoskeletal Remodeling Required for Left-Right Organizer Development.

Cellular proliferation is vital for tissue development, including the Left-Right Organizer (LRO), a transient organ critical for establishing the vertebrate LR body plan. This study investigates cell redistribution and the role of specific progenitor cells in LRO formation, focusing on cell lineage and behavior. Using zebrafish as a model, we mapped all mitotic events in Kupffer's Vesicle (KV), revealing an FGF-dependent, anteriorly enriched mitotic pattern. With a KV-specific fluorescent microtubule (MT) line, we observed that mitotic spindles align along the KV's longest axis until the rosette stage, spindles that form after spin, and are excluded from KV. Early aligned spindles assemble cytokinetic bridges that point MT bundles toward a tight junction where a rosette will initially form. Post-abscission, repurposed MT bundles remain targeted at the rosette center, facilitating actin recruitment. Additional cells, both cytokinetic and non-cytokinetic, are incorporated into the rosette, repurposing or assembling MT bundles before actin recruitment. These findings show that initial divisions are crucial for rosette assembly, MT patterning, and actin remodeling during KV development.

Analysis of Antinuclear Antibody Test Referral Patterns in a Tertiary Care Hospital over Three Years: A Retrospective Observational Study

Introduction: Antinuclear Antibodies (ANA) detection by Human Epithelial Substrate (Hep2) is the recommended screening test for the diagnosis of ANA-Associated Rheumatic Diseases (AARD). ANA is ordered by various specialists in a tertiary care hospital, and a positive ANA result is followed by testing for specific autoantibodies. High pretest probability, pattern type with intensity, and referral departments are key factors determining the autoimmune diagnosis. Aim: To analyse the ANA referral patterns among the various departments and also to estimate the prevalence and clinical significance of rare ANA patterns. Materials and Methods: A retrospective observational study was conducted at Nizam's Institute of Medical Sciences, Hyderabad, a tertiary care hospital, in Hyderabad, Telangana, India where ANA test reports (n=16,994) from various departments over three years (November 2017 to October 2020) were evaluated between November 2022 to December 2022. ANA tests were performed on Hep2 substrate at a 1:100 dilution, and ANA patterns were reported according to the International Consensus on ANA Patterns (ICAP) nomenclature. Statistical analysis of department- wise ANA positivity and fluorescence intensity was conducted, and the final diagnoses of patients with rare ANA patterns (<1%) were noted from clinical records. Fischer's-exact test was used for comparing categorical variables, considering p-value <0.05 as statistically significant. Results: The majority of ANA requests were from the Rheumatology department (5859; 34.5%), followed by nephrology (3132; 18.4%), neurology (1940; 11.4%), general medicine (1646; 9.7%), haematology (1106; 6.5%), and casualty (878; 5.2%), accounting for 85.6% of total referrals. The highest percentage of positivity among ANA referrals was observed in Rheumatology (333; 5.7%), with 58% of positive ANA showing 4+ intensity. No positives were observed from many surgical departments. Rare ANA patterns with a prevalence of less than 1% were observed in 22 patients with mitotic patterns accounting for the majority of rare patterns seen in 11 out of 22 (50%) cases followed by nuclear envelope, rods rings, and nuclear dense fine speckled patterns were observed in six, four, and one patient(s), respectively. The majority of rare ANA patterns had 2+ fluorescence intensity without any associated autoimmune diagnosis. Conclusion: The highest and lowest positivity among ANA referrals were observed in the rheumatology and surgical departments, respectively. Considering the pretest probability of AARD before ordering an ANA test would lead to the optimum utilisation of laboratory services. Mitotic patterns constituted the majority of rare ANA patterns and need to be clinically correlated with antibody titers.

B-131 The Prevalence and Clinical Relevance of the DFS Immunofluorescent Staining Pattern in a Large ANA-positive Cohort

Abstract Background The dense fine-speckled (DFS) nuclear pattern is one of the most common indirect immunofluorescence (IIF) patterns detected during routine antinuclear antibody (ANA) screening. Although various researchers have studied the DFS pattern in recent years, it presents a clinical puzzle since its significance remains elusive. Anti-DFS-70 antibodies occur in healthy individuals with different medical conditions. Unlike the other ANA patterns, they are not associated with systemic autoimmune disease. The present study examined the association of a DFS ANA pattern with the medical conditions of patients who presented to a tertiary care medical center. Methods Retrospective chart review of all laboratory values (January 2020 through June 2022) with ANA- and DFS-positive patterns in patients with ANA tests ordered by various clinicians and compared to the patient diagnosis reported. UMass Human IRB approves this study. Results Of 8885 ANA samples analyzed, 67.8% (6022) were reported negative. Of total positive ANA (2863) samples, 78.6% (2257) had nuclear patterns, 9.1% (260) had mitotic patterns, and 12.1% (346) had cytoplasmic patterns. Of the positive ANA samples, 14.6% (417) had DFS patterns. For samples with DFS pattern, titers ranged from 1:40 to 1:1280; 62.5% were between1:40, and 1:80. Of samples positive for ANA DFS patterns, 76% (317) were from female patients, and 90% (375) were from patients >21 years of age. Among patients with DFS patterns, the most commonly reported diseases were neurological, followed by respiratory, digestive, circulatory, and musculoskeletal (Table 1). Conclusion Clinicians should use a positive ANA with a DFS pattern to indicate pathological phenomena other than autoimmune diseases. Only 3.1% of the patients with DFS patterns have a systemic rheumatic disorder. Clinicians should pay close attention to ANA Patterns, especially DFS staining when using ANA testing.

Standardization of canine meningioma grading: Validation of new guidelines for reproducible histopathologic criteria.

Canine meningiomas are currently graded using the human grading system. Recently published guidelines have adapted the human grading system for use in dogs. The goal of this study was to validate the new guidelines for canine meningiomas. To evaluate the inter-observer agreement, 5 veterinary surgical pathologists graded 158 canine meningiomas following the human grading system alone or with the new guidelines. The inter-observer agreement for histologic grade and each of the grading criteria (mitotic grade, invasion, spontaneous necrosis, macronucleoli, small cells, hypercellularity, pattern loss and anaplasia) was evaluated using the Fleiss kappa index. The diagnostic accuracy (sensitivity and specificity) was assessed by comparing the diagnoses obtained with the 2 grading systems with a consensus grade (considered the reference classification). The consensus histologic grade was obtained by agreement between 4 experienced veterinary neuropathologists following the guidelines. Compared with the human grading alone, the canine-specific guidelines increased the inter-observer agreement for: histologic grade (κ = 0.52); invasion (κ = 0.67); necrosis (κ = 0.62); small cells (κ = 0.36); pattern loss (κ = 0.49) and anaplasia (κ = 0.55). Mitotic grade agreement remained substantial (κ = 0.63). The guidelines improved the sensitivity in identifying grade 1 (95.6%) and the specificity in identifying grade 2 (96.2%) meningiomas. In conclusion, the new grading guidelines for canine meningiomas are associated with an overall improvement in the inter-observer agreement and higher diagnostic accuracy in diagnosing grade 1 and grade 2 meningiomas.

A deep learning self-attention cross residual network with Info-WGANGP for mitotic cell identification in HEp-2 medical microscopic images

BackgroundThe identification of human epithelial type-2 mitotic cell patterns in the indirect immunofluorescence images (IIF HEp-2) is a critical step for autoimmune diseases computer-aided diagnosis (CAD) systems. Recognition of HEp-2 cells in the mitotic phase is clinically vital for validating the HEp-2 samples and assisting in diagnosing specimens with mixed patterns. Typically, mitotic cells are rarely observed in the HEp-2 specimen images, resulting in a significant skewness of the available medical datasets towards the majority of interphase (non-mitotic) patterns. MethodsIn this paper, a deep learning framework is proposed based on a self-attention deep cross-residual network with an efficient generative adversarial network (GAN). The proposed framework consists of two consecutive steps. First, the problem of imbalanced data of minority mitotic against the majority interphase cells is remedied using Info-WGANGP approach in combination with the conventional data augmentation methods. Second, a downstream end-to-end deep learning Att-DCRNet is developed to classify the mitotic and interphase cell patterns of the IIF HEp-2 medical images. A comprehensive experimental study is performed to validate the effectiveness of the proposed framework against other state-of-the-art methods over the public medical dataset of UQ-SNP_HEp-2 Task-3. ResultsThe proposed framework demonstrates competitive classification results attaining a maximum performance of 84.10% F1-score, 84.70% Matthew's correlation coefficient (MCC), and 99.0% balanced class accuracy (BcA), which proves its applicability for automatically supporting an accurate diagnosis decision regarding the HEp-2 mitotic and interphase cell patterns. The source code is available at this link: https://github.com/Anaam-dl/AttDCRNet_with_InfoWGANGP.

Mitogen-induced defective mitosis transforms neural progenitor cells.

Chromosome instability (CIN) with recurrent copy number alterations is a feature of many solid tumors, including glioblastoma (GBM), yet the genes that regulate cell division are rarely mutated in cancers. Here, we show that the brain-abundant mitogen, platelet-derived growth factor-A (PDGFA) fails to induce the expression of kinetochore and spindle assembly checkpoint genes leading to defective mitosis in neural progenitor cells (NPCs). Using a recently reported in vitro model of the initiation of high-grade gliomas from murine NPCs, we investigated the immediate effects of PDGFA exposure on the nuclear and mitotic phenotypes and patterns of gene and protein expression in NPCs, a putative GBM cell of origin. NPCs divided abnormally in defined media containing PDGFA with P53-dependent effects. In wild-type cells, defective mitosis was associated with P53 activation and cell death, but in some null cells, defective mitosis was tolerated. Surviving cells had unstable genomes and proliferated in the presence of PDGFA accumulating random and clonal chromosomal rearrangements. The outcome of this process was a population of tumorigenic NPCs with recurrent gains and losses of chromosomal regions that were syntenic to those recurrently gained and lost in human GBM. By stimulating proliferation without setting the stage for successful mitosis, PDGFA-transformed NPCs lacking P53 function. Our work describes a mechanism of transformation of NPCs by a brain-associated mitogen, raising the possibility that the unique genomic architecture of GBM is an adaptation to defective mitosis that ensures the survival of affected cells.

Rare Antinuclear Antibody Patterns: Relevance in Routine Laboratory Reporting

Introduction: Uncommon patterns on Human Epithelial 2 (Hep 2) substrate during Antinuclear Antibody (ANA) screening by Indirect Immunofluorescence (IIF) microscopy are not routinely reported by many laboratories since their clinical relevance is not well established. Aim: To elucidate rare ANA patterns on Hep 2 and their possible association with clinical presentation. Materials and Methods: A retrospective study was conducted on ANA reports in the duration January 2021 to March 2022 at the Department of Laboratory Medicine, Dr. Sulaiman Al Habib Medical Group, Olaya Medical Complex, Riyadh, Saudi Arabia, to recognise rare ANA patterns. Gold standard method of IIF on Hep 2 was used for screening. Statistical evaluation was done to obtain frequencies of various ANA patterns. Those with frequency of less than 1% were classified as rare patterns. Results: Overall, 4207 consecutive ANA reports were evaluated out of which 1388 were positive and 210 (4.99%) demonstrated rare ANA patterns including nuclear, cytoplasmic and mitotic subtypes. Most commonly encountered among the rare ANA patterns was intercellular bridge (AC 27) with frequency of 0.78% (n=33). Systemic Lupus Erythematosus (SLE) (10/210) was the most often observed clinical association with rare cytoplasmic and mitotic patterns at titer ≥1:160. Conclusion: Uncommon ANA patterns may be useful in initial work-up of autoimmune illness hence, should be routinely reported. Further studies to enlighten the significance of these patterns, analogous antibodies could be of diagnostic relevance in autoimmune and other diseases.

The Anti-Cytoplasmic and Anti-Mitotic Autoantibodies; Are These Antibodies Associated with Diseases?

Examination of antinuclear antibody (ANA) is used in diagnosis of systemic autoimmune diseases, and the indirect immunofluorescence (IIF) assay using HEp-2 cells is the gold standard method. HEp-2 allows the detection of multiple target antigen-directed autoantibodies. The guide “The International Consensus on ANA Patterns (ICAP)”, characterizes the patterns into three groups: nuclear, cytoplasmic, and mitotic. The majority of these are associated with autoimmune diseases, but some are rarely seen in autoimmune diseases or may be associated with conditions other than autoimmune disease. There is no consensus on how to report cytoplasmic and mitotic patterns-negative or positive. We aimed to examine the characteristics of patients that had cytoplasmic or mitotic staining in ANA evaluation by IIF. In our Medical Microbiology Laboratory, 18985 ANA tests of 16940 patients were studied between 01.01.2015-31.12.2019. Cytoplasmic or mitotic pattern was detected in 393 (2.07%) tests belonging to 385 patients. Cytoplasmic patterns suggestive of anti-mitochondrial antibody (AMA), anti-smooth muscle antibody (ASMA), anti-Jo-1 and anti-ribosomal P-protein were not included. The most common patterns were anti-midbody, anti-spindle fibers, and anti-vimentin patterns. There were 66 rheumatology patients that were negative for ANA but had cytoplasmic or mitotic staining. There was no statistically significant difference between the diagnosis and patterns of these patients. We suggest that the ANA should be reported as “negative” in case of cytoplasmic or mitotic pattern unless the term anti-cell antibody is used. It should be noted in the description part of the report in order to distinguish significant cytoplasmic patterns and give an idea for some specific conditions.

Detection of mitotic HEp-2 cell images: role of feature representation and classification framework under class skew.

We propose and analyze a framework to detect and identify the mitotic type staining patterns among different non-mitotic (interphase) patterns on HEp-2 cell substrate specimen images. This is considered as a principal task in computer-aided diagnosis (CAD) of the autoimmune disorders. Due to the rare appearance of mitotic patterns in whole slide/specimen images, the sample skew between mitotic and non-mitotic patterns is an important consideration.We suggest to apply some effective samples skew balancing strategies for the task of classification between mitotic v/s interphase patterns. Another aspect of this study is to consider the morphology and texture-based differences between both the classes that can be incorporated through effective morphology and texture-based descriptors, including the Gabor and LM (Leung-Malik) filter banks and also through some contemporary filter banks derived from convolutional neural networks (CNN).The proposed framework is evaluated on a public dataset and we demonstrate good performance (0.99 or 1 Matthews correlation coefficient (MCC) in many cases), across various experiments. The study also presents a comparison between hand-engineered and CNN-based feature representation, along with the comparisons with state-of-the-art approaches. Hence, the framework proves to be a good solution for the mentioned skewed classification problem.

Gastrointestinal Stromal Tümörlerin Klinikopatolojik Özellikleri ve Ki-67 proliferasyon İndeksinin Prognostik Önemi: Yeni Bir Prognostik Belirteç Olabilir

Objective: The biological behavior of gastrointestinal stromal tumors (GISTs) varies widely and it is diffucult to predict their malignant potential with the current risk classification criterias. Therefore, we aimed to analyse the prognostic importance of Ki-67 LI for estimating survival outcomes in patients with GISTs.
 Methods: For the last 11 years, between 2006 and 2017, who had been treated after surgery were included. A single pathologist re-defined the histologic examples of all cases retrospectively.
 Results: Totally, 104 patients were included in the study. The median follow-up time was 73 months (range; 6 to 148 months). Seven of the 104 patients (7%) had local recurrence, 26 of the 104 patients (25%) had distant metastases and 11 of the 104 patients (11%) died during the follow-up period. The mean OS was 133 (range; 124 to 141) and the mean DFS was 117 (range; 107 to 127) months for patients. The disease progression or recurrence during follow up and increasing age were the significant prognostic factors for OS. Mitotic count, growth pattern, tumor location and Ki-67 LI were the significant prognostic factors for DFS. According to multivariate analyses, the Ki-67 LI was the only prognostic factor for estimating disease progression or recurrence (p=0.04). 
 Conclusion: The most important prognostic factors that affect OS were the age and disease progression or recurrence of disease. Ki-67 LI was the only prognostic factor for estimating disease progression or recurrence. As the follow-up period increases, we think that Ki-67 LI also will affect overall survival.

Comprehensive summary of mitochondrial DNA alterations in the postmortem human brain: A systematic review.

SummaryBackgroundMitochondrial DNA (mtDNA) encodes 37 genes necessary for synthesizing 13 essential subunits of the oxidative phosphorylation system. mtDNA alterations are known to cause mitochondrial disease (MitD), a clinically heterogeneous group of disorders that often present with neuropsychiatric symptoms. Understanding the nature and frequency of mtDNA alterations in health and disease could be a cornerstone in disentangling the relationship between biochemical findings and clinical symptoms of brain disorders. This systematic review aimed to summarize the mtDNA alterations in human brain tissue reported to date that have implications for further research on the pathophysiological significance of mtDNA alterations in brain functioning.MethodsWe searched the PubMed and Embase databases using distinct terms related to postmortem human brain and mtDNA up to June 10, 2021. Reports were eligible if they were empirical studies analysing mtDNA in postmortem human brains.FindingsA total of 158 of 637 studies fulfilled the inclusion criteria and were clustered into the following groups: MitD (48 entries), neurological diseases (NeuD, 55 entries), psychiatric diseases (PsyD, 15 entries), a miscellaneous group with controls and other clinical diseases (5 entries), ageing (20 entries), and technical issues (5 entries). Ten entries were ascribed to more than one group. Pathogenic single nucleotide variants (pSNVs), both homo- or heteroplasmic variants, have been widely reported in MitD, with heteroplasmy levels varying among brain regions; however, pSNVs are rarer in NeuD, PsyD and ageing. A lower mtDNA copy number (CN) in disease was described in most, but not all, of the identified studies. mtDNA deletions were identified in individuals in the four clinical categories and ageing. Notably, brain samples showed significantly more mtDNA deletions and at higher heteroplasmy percentages than blood samples, and several of the deletions present in the brain were not detected in the blood. Finally, mtDNA heteroplasmy, mtDNA CN and the deletion levels varied depending on the brain region studied.InterpretationmtDNA alterations are well known to affect human tissues, including the brain. In general, we found that studies of MitD, NeuD, PsyD, and ageing were highly variable in terms of the type of disease or ageing process investigated, number of screened individuals, studied brain regions and technology used. In NeuD and PsyD, no particular type of mtDNA alteration could be unequivocally assigned to any specific disease or diagnostic group. However, the presence of mtDNA deletions and mtDNA CN variation imply a role for mtDNA in NeuD and PsyD. Heteroplasmy levels and threshold effects, affected brain regions, and mitotic segregation patterns of mtDNA alterations may be involved in the complex inheritance of NeuD and PsyD and in the ageing process. Therefore, more information is needed regarding the type of mtDNA alteration, the affected brain regions, the heteroplasmy levels, and their relationship with clinical phenotypes and the ageing process.FundingHospital Universitari Institut Pere Mata; Institut d'Investigació Sanitària Pere Virgili; Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (PI18/00514).

Endometrial Metaplastic/Reactive Changes Coexistent with Endometrial Hyperplasia and Carcinoma: A Morphological and Immunohistochemical Study.

The aim of this study was to assess the relationship between endometrial metaplastic/reactive changes (EMRCs) and endometrial neoplastic lesions. Twenty cases of “simple” (without architecture complexity) EMRCs coexistent with endometrial malignant/premalignant lesions, twenty cases of neoplasia-unassociated EMRCs, and eight cases of complex metaplastic lesions were assessed by immunohistochemistry. EMRCs coexisted with endometrioid carcinoma (n = 12), atypical endometrial hyperplasia (n = 3), serous carcinoma (n = 2), and clear cell carcinoma (n = 3). Neoplasia-associated EMRCs showed a mean Ki67 labeling index of 12.6% (range 0–30%); with nuclear atypia in 16/20 (80%) cases; diffuse p16 expression in 15/20 (75%) cases; and heterogeneous ER, PR, and vimentin expression. Compared to the associated neoplasia, EMRCs showed a lower Ki67 expression (p < 0.001) and higher p16 expression (p < 0.001). No EMRC case showed mitotic activity, PTEN loss, MMR deficiency, nuclear β-catenin, p53-mutant pattern, Napsin A, or AMACR expression. No significant differences were found between neoplasia-associated and neoplasia-unassociated EMRCs. Complex metaplastic lesions showed a lower Ki67 expression than EMRCs (p = 0.044) and PTEN loss in 5/8 cases, even in the absence of nuclear atypia. In conclusion, neoplasia-associated simple EMRCs may show evident atypia and a worrisome immunophenotype, but no data support their involvement in endometrial carcinogenesis. Architectural complexity appears as a crucial factor to identify precancerous lesions.

Hepatotoxicity of methoxychlor and camel milk restoration

PurposeThe purpose of this paper is to investigate the restoration effect of camel's milk against methoxychlor induced liver toxicity.Design/methodology/approachThe present study was carried out to investigate the restoration effect of camel's milk against methoxychlor induced liver toxicity.FindingsMethoxychlor (MXC) caused a significant increase in serum transaminases (aspartate transaminase and alanine transaminase) and alkaline phosphatase, while MXC induced a significant reduction in total protein and albumin levels. MXC significantly inhibited lipid peroxidation and markedly enhanced glutathione in liver homogenate. Pathological damages as degeneration and coagulative necrosis of hepatocytes were established in liver. Newly formed bile ducteules denotes neoplastic changes in the portal tract with abnormal mitotic pattern were associated with the long-term exposure.Originality/valueThe present study concluded that camel milk treatment may play a protective role against methoxychlor-induced liver damage in rats.

Cancer Stem Cell for Tumor Therapy.

Simple SummaryAlthough many methods have been applied in clinical treatment for tumors, they still always show a poor prognosis. Molecule targeted therapy has revolutionized tumor therapy, and a proper target must be found urgently. With a crucial role in tumor development, metastasis and recurrence, cancer stem cells have been found to be a feasible and potential target for tumor therapy. We list the unique biological characteristics of cancer stem cells and summarize the recent strategies to target cancer stem cells for tumor therapy, through which we hope to provide a comprehensive understanding of cancer stem cells and find a better combinational strategy to target cancer stem cells for tumor therapy.Tumors pose a significant threat to human health. Although many methods, such as operations, chemotherapy and radiotherapy, have been proposed to eliminate tumor cells, the results are unsatisfactory. Targeting therapy has shown potential due to its specificity and efficiency. Meanwhile, it has been revealed that cancer stem cells (CSCs) play a crucial role in the genesis, development, metastasis and recurrence of tumors. Thus, it is feasible to inhibit tumors and improve prognosis via targeting CSCs. In this review, we provide a comprehensive understanding of the biological characteristics of CSCs, including mitotic pattern, metabolic phenotype, therapeutic resistance and related mechanisms. Finally, we summarize CSCs targeted strategies, including targeting CSCs surface markers, targeting CSCs related signal pathways, targeting CSC niches, targeting CSC metabolic pathways, inducing differentiation therapy and immunotherapy (tumor vaccine, CAR-T, oncolytic virus, targeting CSCs–immune cell crosstalk and immunity checkpoint inhibitor). We highlight the potential of immunity therapy and its combinational anti-CSC therapies, which are composed of different drugs working in different mechanisms.

Ультрамикроскопическое строение пейеровых бляшек тонкой кишки крыс после коррекции имунофаном экспериментальной иммуносупрессии

Aim. To study the structure of Peyer’s patches at the ultramicroscopic level after the administration of imunofan against the background of experimentally modeled immunosuppression caused by the administration of cyclophosphamide. Materials and methods. The study was carried out on 36 white mature male rats, divided into 2 groups. The Group 1 included rats, which on the 1st day of the experiment were injected with cyclophosphamide at a dose of 200 mg/kg, and then imunofan according to the scheme on days 2, 4, 6, 8, 10 at a dose of 0.7 μg/kg of body weight. Group 2 was formed from rats, which were injected with an equivalent amount of isotonic sodium chloride solution. Animals of the Group 1 were Taken out from the experiment on the days 7 and 30 after the correction of cyclophosphamide-induced immunosuppression with imunofan. Pieces of Peyer’s patches were isolated and processed according to a standard technique. Ultrathin sections were contrasted in a solution of uranyl acetate and lead citrate according to E. Reynolds and studied under an electron microscope with further photographing. Results. It was found that lymphocytes, in most cases, are tightly adjacent to each other on the days 7 and 30 after the correction of cyclophosphamide-induced immunosuppression with imunofan; in the control group, the stroma is more often located between them. In the nucleus of these cells, heterochromatin is located in clumps and adjoins the nuclear membrane; in the control group, it fills two-thirds of the karyoplasm. The cells with patterns of mitosis, lymphoblasts are often found, macrophages contain large lysosomes on days 7 and 30 of experiment, compared with the control group of rats. In plasmocytes, the granular endoplasmic reticulum occupied up to 2/3 of the cell cytoplasm. The stromal component is well expressed in the control group of animals, compared with the experimental one, and is represented by reticulocytes, reticular fibers and vessels, and fibroblastic reticular cells are also found. Conclusion. In an ultramicroscopic study, the administration of the immunomodulator imunofan against the background of cyclophosphamide-induced immunosuppression promoted an increase in lysosomes in macrophages, lymphoblasts, cells with mitotic patterns, as well as the development of a granular endoplasmic reticulum on days 7 and 30 of the experiment, compared with the control group.

The antinuclear antibody HEp-2 indirect immunofluorescence assay: a survey of laboratory performance, pattern recognition and interpretation

BackgroundTo evaluate the interpretation and reporting of antinuclear antibodies (ANA) by indirect immunofluorescence assay (IFA) using HEp-2 substrates based on common practice and guidance by the International Consensus on ANA patterns (ICAP).MethodParticipants included two groups [16 clinical laboratories (CL) and 8 in vitro diagnostic manufacturers (IVD)] recruited via an email sent to the Association of Medical Laboratory Immunologists (AMLI) membership. Twelve (n = 12) pre-qualified specimens were distributed to participants for testing, interpretation and reporting HEp-2 IFA. Results obtained were analyzed for accuracy with the intended and consensus response for three main categorical patterns (nuclear, cytoplasmic and mitotic), common patterns and ICAP report nomenclatures. The distributions of antibody titers of specimens were also compared.ResultsLaboratories differed in the categorical patterns reported; 8 reporting all patterns, 3 reporting only nuclear patterns and 5 reporting nuclear patterns with various combinations of other patterns. For all participants, accuracy with the intended response for the categorical nuclear pattern was excellent at 99% [95% confidence interval (CI): 97–100%] compared to 78% [95% CI 67–88%] for the cytoplasmic, and 93% [95% CI 86%–100%] for mitotic patterns. The accuracy was 13% greater for the common nomenclature [87%, 95% CI 82–90%] compared to the ICAP nomenclature [74%, 95% CI 68–79%] for all participants. Participants reporting all three main categories demonstrated better performances compared to those reporting 2 or less categorical patterns. The average accuracies varied between participant groups, however, with the lowest and most variable performances for cytoplasmic pattern specimens. The reported titers for all specimens varied, with the least variability for nuclear patterns and most titer variability associated with cytoplasmic patterns.ConclusionsOur study demonstrated significant accuracy for all participants in identifying the categorical nuclear staining as well as traditional pattern assignments for nuclear patterns. However, there was less consistency in reporting cytoplasmic and mitotic patterns, with implications for assigning competencies and training for clinical laboratory personnel.

Rare immunofluorescence patterns of autoantibodies on HEp-2 cells defined by ICAP identify different autoimmune diseases in the absence of associated specificities: a Spanish multicentre study.

ANA are the most extensively used test for the diagnosis of systemic autoimmune diseases. However, testing by indirect immunofluorescence assays (IIFAs) on HEp-2 cells, the gold standard test, is time-consuming and needs expertise. Thus there is a trend to replace it with other automated solid-phase assays directed against specific ANA. Nonetheless, the Hep-2 cell is an autoantigen array and ANA have been classified into 29 types, some of them with no clear association with a specificity to be detected. It is especially in these uncommon patterns where no clinical relationship is found and no antigenic specificity is detected. Here we retrospectively collected clinical data from patients with confirmed uncommon HEp-2 IIFA patterns to search for an associated clinical condition. We conducted an observational retrospective study including 608 patients with organ-specific and non-organ-specific autoimmune diseases (OSADs and NOSADs, respectively) with a confirmed rare pattern of ANA detected by IIFA on HEp-2 cells in the routine practice of the Spanish European Autoantibodies Standardization Initiative laboratories. Inclusion criteria are the existence of a minimum follow-up of 2 years and the availability of clinical data. Nuclear patterns were more frequent in SLE (P = 0.001) and SS (P = 0.001), whereas the cytoplasmic ones were significantly higher in SSc (P = 0.022) and inflammatory myositis (P = 0.016). Mitotic patterns did not show any preferences for a specific disease and 62.7% of them corresponded to the nuclear mitotic apparatus pattern (AC-26). The most frequent NOSADs in patients with the AC-26 pattern were SLE (28.6%), SS (11.9%) and RA (11.9%). The cytoplasmic HEp-2 IIFA patterns were equally distributed in both groups of patients. In the OSAD patients there was no predominant pattern, except for AC-6 in primary biliary cholangitis due to Sp-100 antibodies (P < 0.001). Detection of infrequent ANA might be a unique finding with no disease-associated specificities and could lead to the suspicion of an autoimmune disease.

Anlotinib as a promising inhibitor on tumor growth of oral squamous cell carcinoma through cell apoptosis and mitotic catastrophe

BackgroundOral squamous cell carcinoma (OSCC) has been one of the most malignant cancers in head and neck region. Anlotinib is a tyrosine kinase inhibitor targeting several receptors such as vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR) and c-Kit. Here we investigated whether Anlotinib have any antitumor effect on oral cancer and tried to explore and explain the possible mechanism.MethodsData from The Cancer Genome Atlas and the Gene Expression Omnibus and Gene Expression Omnibus database was collected to analyze the relationship between the expression of vascular epithelial growth factor receptor 2 and the overall survival rate of OSCC. Oral cancer cell lines Cal-27 and SCC-25 were cultured to conduct all the experiments. In vitro experiments such as CCK-8, colony formation, cell cycle assay and cell apoptosis assay were conducted to detect cell proliferation ability and the change of cell phase and apoptosis. Proteins concerning cell cycle and cell apoptosis were visualized via western blot. α-Tubulin were visualized via immunofluorescence to detect cells undergoing mitotic catastrophe.ResultsHigher expression of VEGFR-2 was significantly related to poorer prognosis. Experiment in vitro demonstrated that cell proliferation was significantly inhibited(p < 0.05) after Anlotinib administration and G2/M arrest and apoptosis were both detected in both cell lines. Cycle-related proteins promoting cell cycle progression and proteins related to cell survival were downregulated in Anlotinib group compared to the control group. Cell-death-related biomarker and phosphorylated histone 3 were upregulated in expression in Anlotinib group. Abnormal spindle apparatus was observed in cells undergoing mitotic catastrophe.ConclusionsAnlotinib could exert an antitumor effect on oral cancer cell lines via apoptotic pathway and mitotic catastrophe pattern, presenting a promising potential therapy for patients with OSCC.

Prevalence of autoantibodies to cellular cytoplasmic and mitotic antigens in routine antinuclear antibody reporting: Implementation of international consensus on antinuclear antibodies patterns guidelines

Background: The international consensus on antinuclear antibody (ANA) patterns (ICAP) guidelines recommends that non-nuclear patterns should be reported as positive for ANA. Therefore, a prospective study was planned to investigate the frequency of cytoplasmic and mitotic ANA patterns in routine reporting in a high-throughput lab in a tertiary referral center in Northern India. Materials and Methods: Whole blood samples received were centrifuged and serum was separated within 4 hours of collection. Sera were subjected to indirect immunofluorescence (IIF) assay on HEp-2 cells for ANA determination in a dilution of 1:40, per standardized protocol in our laboratory. IIF staining patterns and intensity were evaluated and the results interpreted. Results: Over a 3-month period, of the 3796 serum samples received for ANA testing, 81 (4.9%) samples were positive for cytoplasmic, 7 (0.4%) for mitotic, and 2 (0.1%) for DNA topoisomerase I (Topo I)-like pattern. The most frequent cytoplasmic fluorescence pattern was dense fine speckled (2%) and the most frequent mitotic pattern was NuMA-like (0.2%). The prevalence of total positive ANA in our study population was found out to be 43.6%. Conclusions: Implementation of ICAP guidelines into routine ANA reporting helped us in detecting important cytoplasmic antibodies such as antimitochondrial antibodies, anti-ribosomal P protein antibodies, anti-Jo-1antibodies, anti-Topo I antibodies and allowed us to introduce more specific and confirmatory tests for the same. The new format for ANA reporting also encouraged clinician-laboratory crosstalk.

Current laboratory and clinical practices in reporting and interpreting anti-nuclear antibody indirect immunofluorescence (ANA IIF) patterns: results of an international survey

BackgroundThe International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP) has recently proposed nomenclature in order to harmonize ANA indirect immunofluorescence (IIF) pattern reporting. ICAP distinguishes competent-level from expert-level patterns. A survey was organized to evaluate reporting, familiarity, and considered clinical value of ANA IIF patterns.MethodsTwo surveys were distributed by European Autoimmunity Standardization Initiative (EASI) working groups, the International Consensus on ANA Patterns (ICAP) and UK NEQAS to laboratory professionals and clinicians.Results438 laboratory professionals and 248 clinicians from 67 countries responded. Except for dense fine speckled (DFS), the nuclear competent patterns were reported by > 85% of the laboratories. Except for rods and rings, the cytoplasmic competent patterns were reported by > 72% of laboratories.Cytoplasmic IIF staining was considered ANA positive by 55% of clinicians and 62% of laboratory professionals, with geographical and expertise-related differences.Quantification of fluorescence intensity was considered clinically relevant for nuclear patterns, but less so for cytoplasmic and mitotic patterns. Combining IIF with specific extractable nuclear antigens (ENA)/dsDNA antibody testing was considered most informative.Of the nuclear competent patterns, the centromere and homogeneous pattern obtained the highest scores for clinical relevance and the DFS pattern the lowest. Of the cytoplasmic patterns, the reticular/mitochondria-like pattern obtained the highest scores for clinical relevance and the polar/Golgi-like and rods and rings patterns the lowest.ConclusionThis survey confirms that the major nuclear and cytoplasmic ANA IIF patterns are considered clinically important. There is no unanimity on classifying DFS, rods and rings and polar/Golgi-like as a competent pattern and on reporting cytoplasmic patterns as ANA IIF positive.