Primate Tissue Research Articles

Primate liver tissue substrate in indirect immunofluorescence diagnostics for patients with dermatitis herpetiformis and celiac disease.

Dermatitis herpetiformis (DH) is a rare autoimmune, polymorphous blistering disorder, characterized by severe itch or burning sensation, which represents the cutaneous manifestation of celiac disease (CD). The current estimation of DH versus CD is around 1:8 and the affected individuals have a genetic predisposition. Pathogenetically, IgA autoantibodies against the epidermal transglutaminase, an essential constituent of the epidermis, cause DH and are reported to develop through cross-reaction with the tissue transglutaminase, with IgA auto-antibodies causing CD. Immunofluorescence techniques allow for a rapid diagnostics of the disease using patient sera. Evaluation of IgA endomysial deposition with indirect immunofluorescence on monkey oesophagus is highly specific, but moderately sensitive, with some operator-dependent variability. Recently, indirect immunofluorescence with monkey liver as a substrate has been proposed as an alternative, well-functioning diagnostic approach with higher sensitivity in CD. The objective of our study was to evaluate whether monkey oesophagus or liver tissue shows advantage for diagnostics in patients with DH, compared to CD. To that end, sera of 103 patients with DH (n=16), CD (n=67) and 20 controls ere compared by 4 blinded experienced raters. For DH, we found a sensitivity of 94.2% for monkey liver (ML) compared to 96.2% in monkey oesophagus (ME), while specificity in ML was superior (91.6% versus 75%) to ME. In CD, ML had a sensitivity of 76.9% (ME 89.1%) and specificity of 98.3% (ME 94.1%). Our data show that ML substrate is well suitable for DH diagnostics.

Brief Communication: Histological Assessment of Nonhuman Primate Brown Adipose Tissue Highlights the Importance of Sympathetic Innervation.

The objective of this study was to functionally analyze the correlation of key histological features in brown adipose tissue (BAT) with clinical metabolic traits in nonhuman primates. Axillary adipose tissue biopsies were collected from a metabolically diverse nonhuman primate cohort with clinical metabolism-related data. Expression of tyrosine hydroxylase (TH), uncoupling protein 1 (UCP1), cluster of differentiation 31 (CD31), cytochrome c oxidase subunit 4 (COX IV), beta-3 adrenergic receptor (β3-AR), and adipose cell size were quantified by immunohistochemical analysis. Computed tomography scans were performed to assess body composition. Tyrosine hydroxylase was negatively correlated with whole body fat mass as a percentage of body weight (p = 0.004) and was positively correlated with the density of UCP1 (p = 0.02), COX IV (p = 0.006), CD31 (p = 0.007), and cell density (p = 0.02) of the BAT samples. Beta-3 adrenergic receptor abundance had a weak positive correlation with COX IV (p = 0.04) in BAT but did not significantly correlate to UCP1 or TH expression in BAT. Our findings highlight that there is a disparity in innervation provided to BAT based on body composition, as seen with the negative association between TH, a marker for innervation, and adiposity. These findings also support the importance of innervation in the functionality of BAT, as TH abundance not only supports leaner body composition but is also positively correlated with known structural elements in BAT (UCP1, COX IV, CD31, and cell density). Based on our observations, β3-AR abundance does not strongly drive these structural elements or TH, all of which are known to be important in the function of brown adipose tissue. In effect, while the role of other receptors, such as β2-AR, should be reviewed in BAT function, these results support the development of safe sympathetic nervous system stimulants to activate brown adipose tissue for obesity treatment.

Single cell multi-omic reference atlases of non-human primate immune tissues reveals CD102 as a biomarker for long-lived plasma cells

In response to infection or immunization, antibodies are produced that provide protection against re-exposure with the same pathogen. These antibodies can persist at high titers for decades and are maintained by bone marrow-resident long-lived plasma cells (LLPC). However, the durability of antibody responses to immunization varies amongst vaccines. It is unknown what factors contribute to the differential longevity of serum antibody responses and whether heterogeneity in LLPC contributes to this phenomenon. While LLPC differentiation has been studied extensively in mice, little is known about this population in humans or non-human primates (NHP). Here, we use multi-omic single-cell profiling to identify and characterize the LLPC compartment in NHP. We identify LLPC biomarkers including the marker CD102 and show that CD102 in combination with CD31 identifies LLPC in NHP bone marrow. Additionally, we find that CD102 is expressed by LLPC in mouse and humans. These results further our understanding of the LLPC compartment in NHP, identify biomarkers of LLPC, and provide tissue-specific single cell references for future studies.

An optimized protocol for the enrichment of small vesicles from murine and non-human primate heart tissue

Over the last few years, the interest in extracellular vesicles (EVs) function has exponentially grown. However, methods for isolating these small vesicles from tissue are still not trivial. Few protocols that allow EV isolation from whole tissue samples, including the heart, are available and they are based on organ perfusion unsing Langendorff method. In this work, aiming at analysing in vivo biology of small EVs, we implemented a simple method to obtain enrichment of these vesicles from murine heart tissue. We tested a titration of Liberase for tissue digestion, which was subjected to differential ultracentrifugation combined with iodixanol cushion and presented the step-by-step procedure of this protocol. Validation was done with Nanoparticle Tracking Analysis, transmission Electron Microscope and Western Blot analysis of EV markers and organelle contaminants. Furthermore, we tested the suitability of the protocol for isolating EVs from heart tissue obtained from a pre-clinical translational non-human primate animal model. Therefore, this protocol should be suitable for isolating vesicle from human heart tissue. Additionally, this method could potentially be applied beyond heart tissue.

P.198 Durable AOC mediated exon 44 skipping in non-human primate muscle tissue and dystrophin protein restoration in DMD patient derived skeletal muscle cells

P.198 Durable AOC mediated exon 44 skipping in non-human primate muscle tissue and dystrophin protein restoration in DMD patient derived skeletal muscle cells

An innovative strategy to identify new targets for delivering antibodies to the brain has led to the exploration of the integrin family.

Increasing brain exposure of biotherapeutics is key to success in central nervous system disease drug discovery. Accessing the brain parenchyma is especially difficult for large polar molecules such as biotherapeutics and antibodies because of the blood-brain barrier. We investigated a new immunization strategy to identify novel receptors mediating transcytosis across the blood-brain barrier. We immunized mice with primary non-human primate brain microvascular endothelial cells to obtain antibodies. These antibodies were screened for their capacity to bind and to be internalized by primary non-human primate brain microvascular endothelial cells and Human Cerebral Microvascular Endothelial Cell clone D3. They were further evaluated for their transcytosis capabilities in three in vitro blood-brain barrier models. In parallel, their targets were identified by two different methods and their pattern of binding to human tissue was investigated using immunohistochemistry. 12 antibodies with unique sequence and internalization capacities were selected amongst more than six hundred. Aside from one antibody targeting Activated Leukocyte Cell Adhesion Molecule and one targeting Striatin3, most of the other antibodies recognized β1 integrin and its heterodimers. The antibody with the best transcytosis capabilities in all blood-brain barrier in vitro models and with the best binding capacity was an anti-αnβ1 integrin. In comparison, commercial anti-integrin antibodies performed poorly in transcytosis assays, emphasizing the originality of the antibodies derived here. Immunohistochemistry studies showed specific vascular staining on human and non-human primate tissues. This transcytotic behavior has not previously been reported for anti-integrin antibodies. Further studies should be undertaken to validate this new mechanism in vivo and to evaluate its potential in brain delivery.

An imaging mass cytometry immunophenotyping panel for non-human primate tissues.

It has recently become clear that spatial organization contributes to cellular function and that expanding our knowledge on cellular organization is essential to further our understanding of processes in health and disease. Imaging mass cytometry enables high dimensional imaging of tissue while preserving spatial context and is therefore a suitable tool to unravel spatial relationships between cells. As availability of human tissue collected over the course of disease or infection is limited, preclinical models are a valuable source of such material. Non-human primate models are used for translational research as their anatomy, physiology and immune system closely resemble those of humans due to close evolutionary proximity. Tissue from non-human primate studies is often preserved large archives encompassing a range of conditions and organs. However, knowledge on antibody clones suitable for FFPE tissue of non-human primate origin is very limited. Here, we present an imaging mass cytometry panel development pipeline which enables the selection and incorporation of antibodies for imaging of non-human primate tissue. This has resulted in an 18-marker backbone panel which enables visualization of a broad range of leukocyte subsets in rhesus and cynomolgus macaque tissues. This high-dimensional imaging mass cytometry panel can be used to increase our knowledge of cellular organization within tissues and its effect on outcome of disease.

Multiple modes of antigen exposure induce clonotypically diverse epitope-specific CD8+ T cells across multiple tissues in nonhuman primates.

Antigen-specific CD8+ T cells play a key role in the host’s antiviral response. T cells recognize viral epitopes via the T cell receptor (TCR), which contains the complementarity-determining region-3 (CDR3), comprising the variable, diversity and joining regions of the TCRβ gene. During chronic simian immunodeficiency virus (SIV) infection of Asian macaque nonhuman primates, tissue-specific clonotypes are identifiable among SIV-specific CD8+ T cells. Here, we sought to determine level of antigen exposure responsible for the tissue-specific clonotypic structure. We examined whether the priming event and/or chronic antigen exposure is response for tissue-specific TCR repertoires. We evaluated the TCR repertoire of SIV-specific CD8+ T cells after acute antigen exposure following inoculation with a SIV DNA vaccine, longitudinally during the acute and chronic phases of SIV, and after administration of antiretrovirals (ARVs). Finally, we assessed the TCR repertoire of cytomegalovirus (CMV)-specific CD8+ T cells to establish if TCR tissue-specificity is shared among viruses that chronically replicate. TCR sequences unique to anatomical sites were identified after acute antigen exposure via vaccination and upon acute SIV infection. Tissue-specific clones also persisted into chronic infection and the clonotypic structure continued to evolve after ARV administration. Finally, tissue-specific clones were also observed in CMV-specific CD8+ T cells. Together, these data suggest that acute antigen priming is sufficient to induce tissue-specific clones and that this clonal hierarchy can persist when antigen loads are naturally or therapeutically reduced, providing mechanistic insight into tissue-residency.

The arteries of the musculoskeletal system of siamangs, and a comparison with other hylobatids, greater apes, and humans.

Limited research on the gross anatomy of the blood vessels has been conducted on hylobatids, or lesser apes, so far. Here, we present a detailed study of the arteries of siamangs (Symphalangus) and compare our findings with data compiled from our previous studies as well as from the literature about other hylobatids, greater apes, and humans. In particular, a three-dimensionalfull-body computed tomography data set of a siamang neonate was analyzed in detail for this study, with notable findings in the head and neck, thorax, upper limb, abdomen and pelvis, and lower limb. Of the 62 arteries that we studied in detail, a total of 20 arteries that have never been described in detail in hylobatids are reported in this study. Key similarities to other apes differing from humans include the existence of a humeral common circumflex trunk and the origination of the dorsalis pedis from the posterior tibial artery or saphenous artery instead of the anterior tibial artery. Similarities to humans differing from other apes include the separation of the lingual and facial arteries and the origination of the profunda brachii from the brachial artery instead of the axillary artery. Our research and broader comparisons, therefore, contribute to knowledge about the soft tissues of hylobatids, other apes, and primates in general and facilitate a better understanding of the anatomical evolution and key differences and similarities among these taxa.

EB3 inhibitor increases chromatin accessibility and reprograms neovascular endothelial cells

In our previous studies, we described a critical role of microtubule‐associated end binding protein 3 (EB3) in regulating pathological calcium release from endoplasmic reticulum (ER) stores through interaction with inositol 1,4,5‐trisphosphate (IP3R3) within endothelial cells. The short cognate peptide we designed based on this interaction blocked VEGF‐evoked ER calcium release and decreased laser‐induced choroidal neovascularization (CNV) in both mouse and non‐human primate models of age‐related macular degeneration. Here we analyzed the global effects of this peptide on choroidal and retinal cells using single nuclei‐RNA (snRNA) and single nuclei‐assay for transposase‐accessible chromatin (snATAC) sequencing of non‐human primate eye tissue. Within the snRNA‐seq transcriptome data, we detected 18 different cell types residing in the retina and choroid using well‐established markers. For the characterization of snATAC‐seq clusters, we performed Pearson correlation of the genes expressed in each cluster of our snRNA‐seq data and the peaks found in each cluster of our snATAC‐seq data. Our snATAC‐seq analysis demonstrated a widespread increase in chromatin accessibility at the promoter region across different cell types. These changes in chromatin accessibility were associated with increased acetylation in lysine 27 of histone 3 (H3K27ac) but not lysine 8 of histone 4 (H4K8ac). Concurrent analysis between our snATAC‐seq and snRNA‐seq data further revealed increased chromatin opening of MEIS2 and PAX6 within our metabolic‐active endothelial cells of neovascularized lesions, leading to activation of downstream transcription factors such as NCOR2, NR2C1, SREBF2, TCF4, TCF12, TEF, and THRA. The activation of this transcriptional program promoted neovascular regeneration and healing of ablated regions within CNV lesion of non‐human primate eye in the treatment group. Taken together, we concluded that EB3 inhibition prevented aberrant angiogenesis through epigenetic reprogramming of injured endothelial cells and through activation of MEIS2 and PAX6’s transcriptional program.

Impact of 6 month conjugated equine estrogen versus estradiol-treatment on biomarkers and enriched gene sets in healthy mammary tissue of non-human primates.

ObjectiveTo identify distinctly regulated gene markers and enriched gene sets in breast tissue of cynomolgus monkeys (Macaca fascicularis) treated for six months with either conjugated equine estrogens (CEE) or estradiol (E2) by analysis of corresponding mRNA levels of genes associated with breast development, carcinogenesis, apoptosis and immune regulation. Additionally, translation of three nuclear markers was analyzed.MethodsRNA from breast biopsies and necropsies was isolated from two independent study trials from Ethun et al. (CEE) and Foth et al. (E2) after 6 month of treatment duration. RNA was subjected to qRT-PCR and MicroArray analysis. Immunohistochemical stainings were performed for the estrogen receptor alpha subunit (ERa), the progesterone receptor (PGR) and the proliferation marker Ki67.ResultsWe identified a total of 36 distinctly enriched gene sets. Thirty-one were found in the CEE treatment group and five were found in the E2 treatment group, with no overlap. Furthermore, two individual genes IGFBP1 and SGK493 were upregulated in CEE treated animals. Additional targeted qRT-PCR analysis of ten specific estrogen-related genes showed upregulation of three genes (TFF1, PGR and GREB1) after CEE treatment, respectively one gene (TFF1) after E2 treatment. Immunohistochemical stains of breast biopsies showed a significant increase in expression of the PGR marker after CEE treatment.ConclusionsIn this study we identified enriched gene sets possibly induced by CEE or E2 treatment in various processes associated with cancer biology and immunology. This preliminary translational data supports the concept that different estrogen types have different effects on healthy breast tissue and may help generate hypotheses for future research.

Characterization of Immune Cells in Oral Tissues of Non-human Primates.

The oral mucosa contains distinct tissue sites with immune niches capable of either immunogenic or tolerogenic responses. However, immune cell compositions within oral mucosal tissues at homeostasis have not been well-characterized in human relevant tissues. Non-human primates (NHP) are a major model for the human immune system and oral anatomy, and therefore improved understanding of NHP oral immune cell populations can provide important insights for studying disease pathologies and developing therapies. Herein, we characterize immune cell types of three sites within the oral cavity (buccal, sublingual, lingual tonsil) sampled by biopsy and cytobrush in pigtail macaques. Tonsil biopsies had more T-cells, dendritic cells (DCs), DC subtypes, and CD4+ T-cells than buccal or sublingual biopsies when normalized by tissue mass. Biopsy proved to collect more immune cells than cytobrushes, however frequencies of CD45+ subpopulations were comparable between methods. Live cells isolated from biopsied tonsils had greater CD45+ leukocyte frequencies (mean 31.6 ± SD 20.4%) than buccal (13.8 ± 4.6%) or sublingual (10.0 ± 5.1%) tissues. T-cells composed more than half of the CD45+ population in sublingual tissue (60.1 ± 9.6%) and the tonsil (54.6 ± 7.5%), but only 31.9 ± 7.2% in buccal samples. CD20+ B-cells composed a greater percentage of CD45+ leukocytes in the tonsil (12.8 ± 9.1%) than buccal (1.2 ± 1.0%) or sublingual tissues (0.8 ± 1.2%). Immune population comparisons are also made between sex and age. These results present an important step for understanding the oral immune environment, oral disease, and site-specific therapy development.

On the relevance of regional collections

Biological collections are the basis of the Earth’s biodiversity knowledge and most of them are regional collections. Here we present two collections from the Federal University of Espírito Santo—Mammal Collection (UFES-MAM) and the associated Animal Tissue Collection (UFES-CTA)—which have been the main repository for mammal specimens collected in Espírito Santo, mostly georeferenced (~ 90%), and available in public databases. Thus, our objective with this essay was to point out the contributions of these collections to the knowledge of the diversity of mammals in the Atlantic Forest. At the same time, we present the contributions they return to society and the scientific community, highlighting the main obstacles and challenges those regional collections face. Despite being regional, UFES-MAM and UFES-CTA stand out nationally for having biological material from a wide variety of species—mainly rodents, marsupials, and bats—also counting on a series of primate tissues collected during the outbreak of wild yellow fever in Southeastern Brazil from 2017 to 2018. These collecting efforts contributed to 42 new mammal species records for Espírito Santo, and 25 species that had already been registered in the state but were listed only in out-of-state collections or reported in literature without voucher specimens. We hope that the information reported here are examples of good practices and increase knowledge and visibility of the rich collection that these regional collections house.

Pathogenesis and Virulence of Chlamydia Trachomatis

The pathogenesis of C. trachomatis disease is a multi-step process that includes: (1) infectivity and exposure to the organism (2) Susceptibility to infection and sickness related to the host's genetic makeup. Recurrence and chronic infections are also common in at-risk teenage and young adult groups. Antibiotic resistance to the primary medications used to treat C. trachomatis is becoming increasingly widespread, even with the correct diagnosis. Chlamydial infection can prevent tumor necrosis factor (TNF)-an-induced physiological apoptosis. Failure to adequately prevent, identify, treat, and remove infection increases the risk of pathogenicity and illness. The plasmid glycoproteins 1–8 (pGP1–8) encode eight open reading frames and most Chlamydia species. In the United Kingdom, there has been a recent increase in the prevalence of such illnesses, whereas, in the Scandinavian nations, there has been a drop, albeit there has been a minor increase in recent years (owing to the development of nucleic acid testing technologies, to some extent). However, it should be noted that reliable monitoring systems and population-based data are Chlamydia trachomatis 4 gitis. Chlamydia trachomatis is made plasma or accessible; moreover, it’s weakened in the vaginal canal of the mouse and nonhuman primate ocular tissue. The plasmid-free organisms ‘in vivo but not in vitro traits were completely mimeographs when pGP3 was inadequate, demonstrating that plasmid-encoded pGP3 is a critical virulence factor in vivo. Moreover, leading to a shortage of cost-effective moment in time tests, including methodologies consistent with strain typing during therapeutic, and the overall degree underlying therapeutic failure is foreign. Those disadvantages were exacerbated because the rest of the genders’ infestations were undiagnosed, allowing continuous silence propagation and developmental defects. The popular medications C. tachometers are becoming more prevalent with proper identification.

Sox-positive cell population in the adult cerebellum increases upon tissue degeneration

Adult neurogenesis is well-described in the subventricular and subgranular zones of the mammalian brain. Recent observations that resident glia express stem cell markers in some areas of the brain not traditionally associated with neurogenesis hint to a possible role in tissue repair. The Bergmann glia (BG) population in the cerebellum displays markers and in vitro features associated with neural stem cells (NSC), however the physiological relevance of this phenotypic overlap remains unclear in the absence of established in vivo evidence of tissue regeneration in the adult cerebellum.Here, this BG population was analysed in the adult cerebellum of different species and showed conservation of NSC-associated marker expression including Sox1, Sox2 and Sox9, in chick, primate and mouse cerebellum tissue. NSC-like cells isolated from adult mouse cerebellum showed slower growth when compared to lateral ventricle NSC, as well as differences upon differentiation. In a mouse model of cerebellar degeneration, progressive Purkinje cell loss was linked to cerebellar cortex disorganisation and a significant increase in Sox-positive cells compared to matching controls. These results show that this Sox-positive population responds to cerebellar tissue disruption, suggesting it may represent a mobilisable cellular resource for targeted strategies to promote tissue repair.

Nonhuman primates\u2019 tissue banks: resources for all model organism research

Biobanks containing tissue and other biological samples from many model organisms provide easy and faster access to ex vivo resources for a wide-range of research programmes. For all laboratory animals, collecting and preserving tissue at post-mortem is an effective way of maximising the benefits of individual animals and potentially reducing the numbers required for experimentation in the future. For primate tissues, biobanks represent the scarcest of these resources but quite possibly those most valuable for preclinical and translation studies.

AEOL 10150 Alleviates Radiation-induced Innate Immune Responses in Non-human Primate Lung Tissue.

To study the molecular and cellular mechanisms of radiation-induced lung injury (RILI) in a non-human primate model, Rhesus macaques were irradiated with lethal doses of radiation to the whole thorax. A subset of the irradiated animals was treated with AEOL 10150, a potent catalytic scavenger of reactive oxygen and nitrogen species. Lung tissues were collected at necropsy for molecular and immunohistochemical (IHC) studies. Microarray expression profiling in the irradiated lung tissues identified differentially expressed genes (DEGs) and pathways important in innate immunity. The elevated expression of cytokines (CCL2, CCL11, IL-8), complement factors (CFB, C3), apoptosis-related molecules (p53, PTEN, Bax, p21, MDM2, c-Caspase 3), and adhesion molecules (fibronectin, integrin β6, ICAM-1) were further studied using real-time PCR, Western blot, or IHC. Oxidative stress and pulmonary inflammatory cell infiltration were increased in the irradiated lungs. Treatment with AEOL 10150 significantly decreased oxidative stress and monocyte/macrophage infiltration. Cytokine/chemokine-induced excessive innate immune response after thoracic irradiation plays an important role in RILI. To our knowledge, this is the first study to highlight the role of cytokine/chemokine-induced innate immune responses in radiation-induced pulmonary toxicity in a NHP model.

Isolating Natural Adeno-Associated Viruses from Primate Tissues with a High-Fidelity Polymerase.

Adeno-associated viruses (AAVs) are advantageous as gene-transfer vectors due to their favorable biological and safety characteristics, with discovering novel AAV variants being key to improving this treatment platform. To date, researchers have isolated over 200 AAVs from natural sources using PCR-based methods. We compared two modern DNA polymerases and their utility for isolating and amplifying the AAV genome. Compared to the HotStar polymerase, the higher-fidelity Q5 Hot Start High-Fidelity DNA Polymerase provided more precise and accurate amplification of the input AAV sequences. The lower-fidelity HotStar DNA polymerase introduced mutations during the isolation and amplification processes, thus generating multiple mutant capsids with variable bioactivity compared to the input AAV gene. The Q5 polymerase enabled the successful discovery of novel AAV capsid sequences from human and nonhuman primate tissue sources. Novel AAV sequences from these sources showed evidence of positive evolutionary selection. This study highlights the importance of using the highest fidelity DNA polymerases available to accurately isolate and characterize AAV genomes from natural sources to ultimately develop more effective gene therapy vectors.

Spectroscopic Measurement of Methylene Blue Distribution in Organs and Tissues of Hamadryas Baboons during Oral Administration

New research on Methylene Blue (MB), carried out in 2020, shows that it can be an effective antiviral drug as part of COVID-19 treatment. According to the research findings, MB has potential as a direct antiviral drug for the prevention and treatment of COVID-19 in the first stages of the disease. However, the MB accumulation by various types of tissues, as well as by immune cells, has not been previously studied. Therefore, the objective of this study was to obtain spectral data on the interstitial distribution of the administered drug in endothelial tissues in primates. The data on interstitial MB distribution obtained by spectroscopic measurement at both macro- and microlevels during oral administration to Hamadryas baboon individuals demonstrate that MB accumulates in mucous membranes of gastrointestinal tract and the tissues of the respiratory, cardiovascular, immune, and nervous systems. Additionally, it was found that MB was present in lung and brain myeloid cells in significant concentrations, which makes it potentially useful for protection from autoimmune response (cytokine storm) and as a tool for the correction of immunocompetent cells’ functional state during laser irradiation. Since the cytokine storm starts from monocytic cells during SARS-CoV-2 cellular damage and since tumor-associated macrophages can significantly alter tumor metabolism, accumulation of MB in these cells provides a reason to conclude that the immune response correction in COVID-19 patients and change in macrophages phenotype can be achieved by deactivation of inflammatory macrophages in tissues with MB using laser radiation of red spectral range.

Internal audit of two antineuronal immunoblots and the role of indirect immunofluorescence in sample screening

Anti-neuronal antibodies are associated with a range of clinical phenotypes that are often preceded by malignancy. Most of these antibodies can be identified by indirect immunofluorescence (IIF) using commercial primate neural tissue slides. The EUROImmun 12 and upgraded RAVO 14 antigen line immunoblots were directly compared to replace a 9 antigen RAVO immunoblot. Initial assessment was with samples with known positive specificities by IIF. The second phase involved parallel testing of routine pooled negative IIF samples as per institutional standard antineuronal testing algorithm. IIF results and clinical correlation were used to arbitrate on discordant immunoblot results. Correlation in known positives was 80%. The EUROImmun immunoblot did not identify disease causing SOX1 and Recoverin antibodies. Routine pooled analysis did not identify discrepancy between the platforms. Assessment of 318 patient samples did not reveal any cases where a diagnosis was correctly identified by line immunoblot and not IIF. A single sample with positive Yo antibodies was identified by both platforms without clinical or IIF correlation, making this a likely false positive. Subsequent to this audit, Westmead ICPMR has migrated to the 14 antigen RAVO line blot for confirmation of positive IIF or for patients with equivocal results only.