Liam Beiglari,¹ Sean Riminton¹ on behalf of the Concord Neuroimmunology Study Group
¹ Concord Hospital Neuroimmunology Clinic

Paraneoplastic neurological syndromes (PNS) are rare, increasingly reported, and are on the differential diagnosis of a range of neurological presentations. A 2018 paper by Ebright¹ identified a high false positive rate associated with panel testing of autoantibodies for PNS. They identified the “unintended consequences” of testing for PNS including waste and clinical harm. We sought to explore the unintended consequences of these assays in the Australian context by determining the specificity of positive results.

Consecutive results for PNS testing ordered by the Concord Neuroimmunology Service between January 2020 and December 2023 were extracted from the RPA immunopathology laboratory. This included a line blot immunoassay (LIA) (Euroimmun, Paraneoplastic 12 Ag, Lübeck, Germany ) which tests 12 distinct antigen specificities and indirect immunofluorescence (IIF) (Inova Diagnostics, San Diego, USA) on primate brain. Sixty seven positive antigen specificities were compared to clinical data. Duplicates and IIF without positive LIA were excluded from the final analysis.

The final data will be presented in the presentation although preliminary data suggests a high rate of false positives. These data will be used for generating recommendations for amendments to clinical laboratory algorithms in reporting PNS testing.

References:

1.      Ebright MJ, Li S-H, Reynolds EL, Burke JF, Claytor BR, Grisold A, Banerjee M, Callaghan B, Callaghan BC. Unintended consequences of Mayo paraneoplastic evaluations. Neurology 2018;91.

Bronte Jeffrey¹, Marisa Alves¹, Steven Le¹, Elizabeth Foley¹, Samuel Breit^1,2,3 ,William Sewell^{1, 2,4}

¹Department of Immunopathology, Sydpath, St Vincent’s Hospital, Sydney, Australia

²School of Medicine, University of New South Wales, Sydney, Australia

³St Vincent’s Centre for Applied Medical Research (AMR), St Vincent's Hospital. Sydney

⁴The Garvan Institute of Medical Research, Sydney, Australia

Background: Diagnosis of mature T cell neoplasms by flow cytometry is challenging. Historically, Vbeta antibodies have been used to assess T cell clonality. This approach,  however,  has lacked sensitivity, is costly and time consuming. More recently, TRBC1 and TRBC2 antibodies which target mutually exclusive isoforms of the constant beta chain of the T cell receptor have been introduced as an alternative means of clonality assessment. These antibodies have the potential to simplify T cell analysis.

Methods: Specimens referred for flow cytometry were simultaneously analysed with TRBC1 alone and combination TRBC1/TRBC2. 

Results: 139 specimens were analysed for clonal T cell populations. This included 106 control specimens, 16 samples in which a T cell population of uncertain significance (T-CUS) was reported and 17 samples with confirmed T cell neoplasm.  All 17 specimens with confirmed T cell neoplasm were clonal using TRBC1 alone and combination TRBC1/TRBC2 analysis. Overall, prevalence of T-CUS was high with clones identified in 33 (32%) of control specimens.  Furthermore, use of the TRBC1/TRBC2 combination clarified analysis and 11 T-CUS specimens had different clonality using the combination compared to TRBC1 alone.

Conclusion: Combination  TRBC1/TRBC2 antibody in T cell analysis increases certainty in reporting clonal populations compared to TRBC1 alone. 

Karrnan Pathmanandavel¹, Adrian Lee¹, Maria Dela Cruz¹, Suzanne Culican¹, David Campbell¹, Mark Acebes¹, Jonathan Emerson¹, Lawrence Ong¹, David Brown¹, Sarah Sasson¹, Ming Wei Lin¹, Lucinda Berglund¹

¹Department of Immunology and Immunopathology, ICPMR, Westmead Hospital, Sydney, Australia

Proliferative Glomerulonephritis with Monoclonal Immune Deposits (PGNMID) is a renal disease characterised by monoclonal deposition of immunoglobulins, often IgG3 subclass restricted, in the glomerulus. This entity is heterogeneous in its presenting features, demographics, and clinical outcome – with occasional cases of spontaneous remission but 20% progressing to end-stage kidney disease. Interestingly, only a minority (~ 30%) of patients have detectable circulating paraprotein or a clonal population on flow cytometry. Thus, the pathogenesis of PGNMID is unclear, complicating the choice of B-lineage depleting therapy (e.g., rituximab vs. bortezomib). 

We have identified a surprisingly high frequency of PGNMID cases where the light chain clonality observed on renal direct immunofluorescence is opposite to that seen on other studies. This highlights the need for a holistic approach to diagnosis with correlation between multiple test modalities, and particularly the value added by IgG subclass staining. These cases have also suggested novel research approaches to better understand the pathogenesis of this enigmatic entity.  

F. Kakar^1,2, A. Fontes⁵, D. McDonald¹, JDE.Parratt⁵, MW. Lin*^1,2,3,4 and D.A. Brown*^1,2,3,4, 

 

¹ Department of Immunopathology, ICPMR, NSW Health Pathology, Westmead Hospital, Sydney

²Faculty of Medicine and Health Sciences, University of Sydney 

³ Department of Clinical Immunology, Westmead Hospital, Westmead, Sydney

⁴ Centre of Immunology and Allergy Research, Westmead Institute of Medical Research, Westmead, Sydney

⁵Neurology Department, Royal North Shore Hospital, Sydney, NSW

 

Aim: Validation of serum neurofilament light chain (sNFL) by Single Molecular Array (SiMOA) technology.  

 

Introduction: sNFL is an ultrasensitive diagnostic, monitoring and prognostication biomarker of neuronal damage in neurodegenerative disorders, allowing measurement of sNfL values in picograms/ml.

 

Method: NfL levels from 89 healthy males and females ages 20-89 years were measured to establish age partitioned reference range based on existing literature ^[1]. These were compared to 20 multiple sclerosis (MS) patients with high and 20 with low disease activity scores. Further studies included precision testing, batch to batch variation, fridge stability and freeze thaw cycle effects on serum samples. Inter-laboratory quality control testing was also performed.

 

Results: Age partitioned normal reference levels were established. Comparison of diseased samples with age matched controls revealed a significant relationship between sNfL levels and disease activity (p-value of <0.0001). Precision testing showed a maximum intra-laboratory CV of 15%. Stability studies indicated serum samples remain stable for up to 2 weeks in the fridge and 2 freeze thaw cycles. Batch to batch variation, intra and inter-laboratory comparisons were satisfactory.  

 

Conclusion: SiMOA technology reliably measures sNfL levels, meeting NATA accreditation standards at ICPMR, NSW Health Pathology. 

 

 

1.       Hviid, C.V.B., Knudsen, C.S. and Parkner, T. (2020). Reference interval and preanalytical properties of serum neurofilament light chain in Scandinavian adults. Scandinavian Journal of Clinical and Laboratory Investigation, 80(4), pp.291–295. 

Adrina Varda^1
1ACT Pathology 

Pernicious anemia is predominantly a haematological manifestation of chronic autoimmune gastritis that ultimately leads to gastric atrophy and vitamin B12 deficiency. The development of these features are due to the presence of autoantibodies. Specifically, autoantibodies to gastric parietal cells are sensitive but not specific for pernicious anemia. The converse applies to intrinsic factor autoantibodies, with incidence noted to increase with disease progression. The decision to undertake both autoantibody tests depends on the laboratory’s algorithm, noting available evidence in literature and the tests requested. Currently at ACT Pathology, autoantibodies to intrinsic factor are sent away to an external laboratory where ELISA is employed. There is no gold standard or standardised platform; other methods employed include CLIA and EliA. This quality improvement project mainly aims to compare these 3 platforms with the view of introducing the most fit-for-purpose and concordant assay. Preliminary data has demonstrated that there is 100% concordance between the external laboratory ELISA results compared to our ELISA (14 samples, Cohen’s kappa 1) and CLIA (25 samples, Cohen’s kappa 1). This is also the case when direct comparisons were made between our ELISA and CLIA. Further analysis of samples will be made in addition to testing the performance of EliA.