Background C3 glomerulopathy is an uncommon form of complement-mediated glomerular disease marked by dysregulation of the alternative complement pathway, sometimes associated with monoclonal light chain deposits driven by a small monoclonal plasma cell or B-cell clone. Digital ischaemia (acral necrosis) is a rarely reported extra-renal manifestation in this setting. With no data on effective therapy for refractory cases, novel complement inhibitors such as pegcetacoplan are a potential therapeutic option. Case presentation We report the case of a 61-year-old man who presented with proteinuria, preserved renal function, and digital ischemia that repeatedly relapsed leading to autoamputation of ten fingers despite standard immunosuppression, plasma exchange and myeloma therapy (CyBorD). Investigations identified a 4 g/L serum IgG lambda monoclonal protein. Renal histopathology confirmed C3-dominant lambda restricted deposits with subendothelial and mesangial localisation. Serum testing found reduced AH100, elevated Factor H and soluble C5b-9, with normal C3 and C4. In the setting of ongoing digital ischemia with threatened toe autoamputations and worsening proteinuria, the patient was commenced on pegcetacoplan therapy. Renal parameters stabilised (proteinuria resolved), and the ischaemic lesions gradually resolved with revascularisation of the toes. No infectious complications have occurred to date with 20 months follow up. Conclusions This is the first reported case of pegcetacoplan achieving clinical reversal of digital ischemia and renal stabilisation in a patient with refractory C3 glomerulopathy, monoclonal gammopathy and severe digital ischaemia. This case underscores the potential utility of targeted complement blockade in severe, extra-renal manifestations of C3 glomerulopathy.

Multiparametric flow cytometry (MFC) enables rapid and sensitive quantification of measurable residual disease (MRD) in patients with acute myeloid leukaemia (AML), providing valuable prognostic information and guiding post-remission therapeutic strategies. Given the clinical utility of MFC AML MRD assessment and its inherent technical and analytical complexity, inter-laboratory standardisation is essential to ensure consistency of practice, diagnostic accuracy and reproducibility of results. However, limited options for external quality assessment exist. Representatives from all diagnostic laboratories in Australia and New Zealand currently performing AML MRD testing were invited to attend an in-person workshop to review site-specific practices and develop harmonisation processes. All six participating laboratories demonstrated a high level of concordance with respect to pre-analytical testing; however, greater variation was observed in post-analytical reporting, and a number of consensus recommendations were formulated for harmonisation. Ongoing meetings have also been established to promote continued sharing of expertise between AML MRD laboratories, align analytical strategies and enhance assay validation in accordance with regulatory requirements. These collaborative efforts provide guidance to existing MRD laboratories and those seeking to establish a new MRD service, facilitating sustainable provision of a high-quality regional MFC AML MRD testing network to meet current and anticipated increases in clinical demand.

The E3 ubiquitin ligase Casitas B-lineage lymphoma (CBL) promotes positive selection and antigen responses in mouse T lymphocytes by ubiquitinating ZAP70. Conversely, mouse CBL and CBL-B mutually redundantly regulate SYK ubiquitination and B cell receptor signaling. Here we studied individuals with somatically homozygous CBL loss-of-function variants in leukocytes. Human CBL is largely redundant for the development and function of human T cells. Conversely, B cell development is altered at the immature stage, with a tenfold increase in transitional cells, enhanced survival of autoreactive clones and impaired tolerance manifested by autoantibody production. B cell maturation is intrinsically impaired by reduced apoptosis and dysregulated B cell receptor signaling. CBL deficiency impairs humoral immunity by limiting memory B cell formation and reducing class switching and somatic hypermutation. Consequently, antigen-specific B cell generation and adaptive immune memory are disrupted, predisposing individuals to infection. Human CBL is critical for B cell development and function but redundant for T cell biology.

BACKGROUND:Fine-needle aspiration biopsy (FNAB) of lymph nodes is a widely used method for evaluating lymphadenopathy. FNAB offers general advantages of rapid turnaround time, low cost and minimal morbidity, and more specific advantages in various clinical situations, such as deeply located lymph nodes or patients with significant comorbidities. The FNAB sample can be utilized for a wide range of ancillary tests, including microbiological studies, immunocytochemistry for primary and metastatic neoplasms and flow cytometry immunophenotyping in cases of lymphoid-rich samples, where there is a suspicion for lymphomas. SUMMARY:The increasing application of FNAB in lymph node pathology has led to the development of a standardized reporting system, formalized in the World Health Organization (WHO) Reporting System for Lymph Node, Spleen and Thymus Cytopathology (WHO System). This system is equally applicable to lymph node, spleen and thymus; however, this article focuses on lymph nodes. The WHO System was established through a joint project of the WHO, the International Agency for Research on Cancer (IARC) and the International Academy of Cytology (IAC) and is structured into five diagnostic categories: inadequate/insufficient/non-diagnostic, benign, atypical, suspicious for malignancy, and malignant. The WHO System provides a standardized and reliable means of categorizing various lymph node lesions based on cytopathology findings and enables pathologists to make more accurate and reproducible diagnoses, thereby improving clinical management and treatment decisions. Integrating cellular morphology and clinical-imaging data help distinguish benign from malignant lesions, significantly reducing diagnostic variability. The primary goal was to reduce diagnostic uncertainty and improve patient outcomes through greater consistency and clarity in lymph node cytopathology reports. The WHO System emphasizes the use of rapid on-site assessment (ROSE) to improve diagnostic accuracy and reduce the need for additional diagnostic procedures. The risk of malignancy (ROM) varies by diagnostic category, with higher risks of malignancy in the "Suspicious for malignancy" and "Malignant" categories. The system also includes recommendations for ancillary tests and performance of additional biopsies when further clarification is needed. The WHO System represents a significant advancement in the standardization of lymph node, spleen, and thymus cytopathology, facilitating interdisciplinary communication and improving risk stratification. However, diagnostic challenges remain, particularly in managing inadequate samples and interpreting atypical lesions, necessitating a multidisciplinary approach that integrates clinical, imaging, ancillary testing and, in some cases, core needle, or excision biopsy material. KEY MESSAGES:The WHO System serves as a crucial tool for refining the diagnosis of the broad range of inflammatory, infectious, metastatic, and lymphomatous processes in lymph node pathologies. In addition, it is suitable for high income as well as most obviously low- and middle-income countries leading to optimizing therapeutic decision-making.

BACKGROUND:The International Academy of Cytology in collaboration with the International Agency for Research on Cancer has developed a standardized World Health Organization Reporting System (WHO System) for the cytopathology of the lymph nodes, spleen, and thymus. Fine-needle aspiration biopsy (FNAB) of the spleen and thymus, performed under ultrasound or computed tomography guidance, constitutes a minimally invasive, well-tolerated, and cost-effective diagnostic technique. Despite a limited number of published studies on the diagnostic accuracy of FNAB in these organs, it remains a valuable tool particularly when combined with rapid on-site evaluation and ancillary techniques, such as immunophenotypic analysis by flow cytometry or immunocytochemistry and molecular testing. SUMMARY:The WHO System categorizes thymic and splenic cytopathology into five diagnostic categories: "Inadequate/Insufficient/Nondiagnostic," "Benign," "Atypical," "Suspicious for malignancy," and "Malignant." This standardized approach aims to enhance diagnostic accuracy, provide risk assessment, and facilitate clinical decision-making. However, applicability of FNAB in the thymus and spleen is still debated, particularly concerning sample adequacy, diagnostic accuracy, and potential complications such as hemorrhage, especially for splenic FNAB. Thymic FNAB is primarily used for diagnosing thymic hyperplasia, thymoma, thymic carcinomas, and lymphomas. However, distinguishing these entities and their subtypes is challenging. Splenic FNAB is valuable in evaluating splenomegaly, infections, lymphomas, and other neoplasms, particularly in staging and monitoring disease progression. While using FNAB to evaluate these organs is not yet a widely adopted standard practice, its role in minimizing unnecessary surgical interventions and guiding therapeutic strategies is being increasingly recognized. KEY MESSAGES:The WHO System for the thymus and spleen seeks to establish a harmonized, evidence-based framework for cytopathological diagnosis, incorporating key diagnostic criteria, malignancy risk assessment, and standardized reporting protocols. Future research is needed to refine and develop diagnostic role of FNAB, enhance its integration with advanced molecular techniques, and optimize its use in personalized medicine.

Aims/Purpose: To determine the diagnostic yield and pathological entities in a retrospective cohort of diagnostic vitrectomy patients. Methods: A retrospective cohort of diagnostic vitrectomy samples submitted to a tertial referral hospital pathology laboratory was identified by database search. The specimen submission protocol for this cohort consisted of two components: (i) intraoperative preparation of a neat vitreous aspirate as well as submission of the casette washings in RPMI and (ii) laboratory triage of each sample for microbiology culture, flow cytometry, cytological assessment and cell block preparation. Results: A total of 133 diagnostic vitrectomy samples were submitted between November 2015 and January 2022 (mean age 74 years, 0.8 M:F). A diagnostic report resulted in 132 (99.2%) of cases. Benign diagnoses (infectious and non‐infectious vitritis, degenerative or reactive changes, haemorrhage) were found in 88 of the cases (66.1%), while 20 cases (15%) were malignant. Of the malignant diagnoses, 18 (13.5%) were vitreoretinal lymphoma (17 diffuse large B cells; 1 T cell) by cytology and flow cytometry. The other two malignant diagnoses consisted of metastatic adenocarcinoma and metastatic cutaneous melanoma. 24 cases (18%) were classified as atypical, consisting of a lymphocytosis without demonstrable clonality by flow cytometry. Conclusions: This intraoperative and laboratory protocol maximizes the sample for all diagnostic tests required to discern infectious, inflammatory and malignant causes of increased vitreous cellularity. Importantly, the protocol allows for immunophenotyping of vitreoretinal lymphoma by flow cytometry and immunohistochemistry to WHO classification standards, essential for treatment and surveillance decision‐making.

AIMS:The recent widespread use of electronic health records (EHRs) has opened the possibility for innumerable artificial intelligence (AI) tools to aid in genomics, phenomics, and other research, as well as disease prevention, diagnosis, and therapy. Unfortunately, much of the data contained in EHRs are not optimally structured for even the most sophisticated AI approaches. There are very few published efforts investigating methods for recording discrete data in EHRs that would not slow current clinical workflows or ways to prioritise patient characteristics worth recording. Here, we propose an approach to identify and prioritise findings (phenotypes) useful for differentiating diseases, with an initial focus on relatively common small B-cell lymphomas. MATERIALS AND METHODS:A website enabling crowd-sourced recording of diseases and phenotypes was developed. An expert committee in the field of B-cell lymphomas standardised phenotype terminology for use in digital resources, and select terms were included in the Human Phenotype Ontology (HPO). A total of 100 patient lymph node biopsy samples were evaluated, and phenotypes were recorded as discrete data. Bayesian networks (BNs) were developed based on these data, and their diagnostic accuracy and ability to prioritise these phenotypes for inclusion in EHRs were assessed. RESULTS:Out of 146 phenotypes identified from the website as potentially useful for differentiating four different lymphomas from each other and from benign lymph nodes, 70-75 were included in BNs. The diagnostic accuracy of different naïve BNs was 96.3% for non-marginal zone lymphoma cases and 50% for marginal zone lymphoma cases when all of the included phenotypes were used and 93.8% for non-marginal zone lymphoma cases and 27.5% for marginal zone lymphoma cases when only 15 phenotypes were included in the BNs. CONCLUSION:This pilot provides a starting point for systematic improvement and a dataset for comparing related approaches.

Cellular immunity, mediated by antigen-specific T cells, is a key component of host control of many commensal and infectious organisms. However, few clinical assays directly measure antigen-specific T cells. Zaunders et al. developed the OX40 system as a robust and practical method to address this need.1 The assay, performed on whole blood without the need for prior processing, utilises flow cytometric quantification of surface antigens upregulated in response to incubation with antigen peptide pools.

Background: αβ T cells express either T cell receptor (TCR) β constant region 1 (TRBC1) or TRBC2. Flow cytometric quantification of TRBC1 expression enables rapid detection of T cell clonality in haematological malignancy and is being widely implemented.1,2 Unexpectedly, we identified clonal CD8 T cell populations in >75% of bone marrow samples tested using this method. We hypothesised that these CD8 populations may be MAIT cells, innate-like T cells with a semi-invariant TCR repertoire that are enriched in various tissue compartments.

Background: Recent studies have shown that mepolizumab is an effective treatment in the management of chronic rhinosinusitis with nasal polyposis (CRSwNP). Defining which patients will benefit from this treatment is essential in guiding its role in the multimodal approach to CRSwNP and eosinophilic chronic rhinosinusitis (eCRS). The aim of this study is to define the clinical and disease features of those patients who report benefit from therapy compared to non-responders. Methods: A prospective phase 2 clinical trial was undertaken with a single -arm of non -blinded patients with open label therapy with mepolizumab (trial registration ID ACTRN12618000113257) from May 2019 to November 2020. Patients underwent treatment with mepolizumab 100 mg 4-weekly for 6 months. Data was collected at baseline and at 4, 8, 12, 16, 20 and 24 weeks of treatment, as well as 4 months post their last mepolizumab dose. Data collected included patient demographics, blood eosinophil count (cells/L), tissue histopathology outcomes [inflammation severity, type, eosinophil density (cell/HPF)], functional outcomes [endoscopic findings, nasal nitric oxide (nNO), fractional exhaled nitric oxide (FeNO)] and patient reported outcome measures [including Sino-Nasal Outcome Test 22-item (SNOT-22), Asthma Control Questionnaire 5-item (ACQ-5), nasal obstruction visual analogue scores (VAS) and nasal function VAS]. Responders were defined as those patients having a >= 1 improvement in a 13 -point ordinal overall nasal function score. Results: Twenty patients were assessed (age 47.7 +/- 14.5 years and 50% female). All patients had prior sinus surgery (4.1 +/- 3.5 years) and all had comorbid asthma. Fourteen patients (70%) were classed as responders to mepolizumab therapy. Responders were associated with higher baseline symptom burden: SNOT-22 (51.3 +/- 17.3 vs. 30.7 +/- 19.1, P=0.03), ACQ-5 (2.3 +/- 1.2 vs. 1.2 +/- 0.5, P=0.03) and lower baseline nNO (300.4 +/- 169.7 vs. 645.0 +/- 318.7 parts per billion, P=0.005). Within 3 months following cessation of mepolizumab, there was a deterioration of clinical outcomes including blood eosinophils, tissue eosinophils, SNOT-22 and ACQ-5. Conclusions: In eCRS, responders were likely to have more severe symptomatic disease at baseline and lower nNO. Cessation of mepolizumab is associated with deterioration of both objective and subjective markers.

Flow cytometry (FCM) is widely used in the diagnosis of mature B‐cell neoplasms (MBN), and FCM data are usually consistent with morphological findings. However, diffuse large B‐cell lymphoma (DLBCL), a common MBN, is sometimes not detected by FCM. This study aimed to explore factors that increase the likelihood of failure to detect DLBCL by FCM.

Background Central compartment atopic disease (CCAD) and eosinophilic chronic rhinosinusitis (eCRS) are two clinical phenotypes of primary diffuse type 2 chronic rhinosinusitis (CRS) defined in the European Position Paper on Rhinosinusitis 2020 classification. Currently, the distinction between these subtypes relies on phenotypic features alone. Objective This study aimed to investigate whether eosinophil activation differed between CCAD and eCRS. Methods A cross-sectional study was conducted of adult patients presenting with CCAD and eCRS who had undergone functional endoscopic sinus surgery. Routine pathology results were obtained from clinical records. Eosinophils were counted on haematoxylin and eosin-stained formalin-fixed paraffin-embedded sinonasal tissue. Eotaxin-3, eosinophil peroxidase and immunoglobulin E levels were assessed using immunohistochemistry. Results 38 participants were included (51.7 +/- 15.6 years, 47.4% female), of whom 36.8% were diagnosed with CCAD and 63.2% with eCRS. The eCRS group was characterised by older age (55.8 +/- 16.3 vs 44.5 +/- 11.8 years, p = 0.029), and on histology exhibited a higher degree of tissue inflammation (tau(b) = 0.409, p = 0.011), greater proportion of patients with >100 eosinophils/high power field (87.5% vs 50%, p = 0.011), and higher absolute tissue eosinophil count (2141 +/- 1947 vs 746 +/- 519 cells/mm(2), p = 0.013). Eotaxin-3 scores were higher in the eCRS group (5.00[5.00-6.00] vs 6.00[6.00-6.75], p = 0.015). Other outcomes were similar. Conclusions Eosinophil and eotaxin-3 levels were elevated in eCRS compared with CCAD, suggesting a greater degree of eosinophil stimulation and chemotaxis. Patients with CCAD were younger. Future investigation and biomarkers may better distinguish CRS subpopulations.

A 78-year-old Caucasian man with history of long-standing HIV well managed on long term antiretroviral therapy, presented to the emergency department with dry cough and shortness of breath of three weeks duration. He had a massive left sided pleural effusion requiring drainage. Flow cytometry analysis of the fluid demonstrated very dim CD45, positive for CD38 and CD138, and aberrant T lineage markers. The cytology was highly cellular with a lymphocyte population staining for plasma cells markers, HHV8 and aberrant T lineage markers. The combination of flow cytometry, cytology, bone marrow analysis, and imaging led to the final diagnosis of primary effusion lymphoma (PEL). The association between HIV and PEL is recognised. However, this case is unusual in that the patient had long-standing well managed HIV infection. The case suggests that other factors such as immune senescence may play a role in the development of PEL.

Heterozygous loss-of-function (LOF) mutations in PIK3R1 (encoding phosphatidylinositol 3-kinase [PI3K] regulatory subunits) cause activated PI3Kδ syndrome 2 (APDS2), which has a similar clinical profile to APDS1, caused by heterozygous gain-of-function (GOF) mutations in PIK3CD (encoding the PI3K p110δ catalytic subunit). While several studies have established how PIK3CD GOF leads to immune dysregulation, less is known about how PIK3R1 LOF mutations alter cellular function. By studying a novel CRISPR/Cas9 mouse model and patients' immune cells, we determined how PIK3R1 LOF alters cellular function. We observed some overlap in cellular defects in APDS1 and APDS2, including decreased intrinsic B cell class switching and defective Tfh cell function. However, we also identified unique APDS2 phenotypes including defective expansion and affinity maturation of Pik3r1 LOF B cells following immunization, and decreased survival of Pik3r1 LOF pups. Further, we observed clear differences in the way Pik3r1 LOF and Pik3cd GOF altered signaling. Together these results demonstrate crucial differences between these two genetic etiologies.

Background Biologic therapies such as mepolizumab and benralizumab are currently utilised in the treatment of eosinophilic asthma, and are emerging in the management of eosinophilic chronic rhinosinusitis (eCRS). These biologics inhibit the interaction of IL-5 with its receptor, thus impairing cytokine signalling and eosinophil inflammation. Mepolizumab does so by targeting IL-5, whereas benralizumab targets the α chain of the IL-5 receptor. This study compares the sinonasal tissue response to anti-IL-5 biologic therapies in patients with eCRS. Methods A cross-sectional study of adult eCRS patients who had completed at least 2 cycles of biologic therapy and underwent endoscopic sinus surgery as part of their management were included. Sinonasal mucosal tissue biopsies were obtained intraoperatively and assessed with structured histopathological examination. Comparisons of tissue histopathology outcomes following treatment with mepolizumab or benralizumab were performed. Results 18 patients (age 49.6 ± 14.2 years, 47% female, 100% co-morbid asthma) were included in this study, comprising 10 patients managed with mepolizumab and 8 patients managed with benralizumab. Even after mepolizumab, the tissue had predominantly eosinophilic inflammation compared to benralizumab (90% v 0%, p < 0.01), which demonstrated a greater lymphoplasmacytic inflammation (10% v 75%, χ2(2) = 14.53, p < 0.01). Compared with benralizumab, mepolizumab had increased tissue eosinophil count (100% v 37.5% >10 eosinophils/HPF, τb = −8.47, p < 0.001) and more severe subepithelial oedema (80% v 37.5% severe, τb = −2.37, p = 0.02). Conclusion Tissue histopathologic outcomes reflect the differing mechanism of action of mepolizumab and benralizumab in eCRS. Further analysis at the tissue level will provide further information to guide application of mAbs in type 2 inflammatory diseases.

The upcoming 5th edition of the World Health Organization (WHO) Classification of Haematolymphoid Tumours is part of an effort to hierarchically catalogue human cancers arising in various organ systems within a single relational database. This paper summarizes the new WHO classification scheme for myeloid and histiocytic/dendritic neoplasms and provides an overview of the principles and rationale underpinning changes from the prior edition. The definition and diagnosis of disease types continues to be based on multiple clinicopathologic parameters, but with refinement of diagnostic criteria and emphasis on therapeutically and/or prognostically actionable biomarkers. While a genetic basis for defining diseases is sought where possible, the classification strives to keep practical worldwide applicability in perspective. The result is an enhanced, contemporary, evidence-based classification of myeloid and histiocytic/dendritic neoplasms, rooted in molecular biology and an organizational structure that permits future scalability as new discoveries continue to inexorably inform future editions.