Journal of Immunological Methods

BackgroundTraditional immunohistochemistry (IHC) with chromogen detection has limited multiplex capacity, detecting at most 4 protein markers per tissue section simultaneously, thereby restricting comprehensive spatial analysis of valuable human biopsies. We developed and validated a robust serial IHC (sIHC) staining method to detect multiple antigens on a single kidney biopsy slide, maximizing data yield for diagnosing and studying complex kidney diseases. MethodsFormalin-fixed, paraffin-embedded kidney biopsy sections were subjected to repeated IHC/imaging cycles with antibody removal using an optimized sodium dodecyl sulfate-glycerol buffer stripping protocol. Images were then co-registered, and analysis was performed using a variety of methodologies, including color deconvolution, cell segmentation, and spatial clustering. ResultsThis optimized sIHC method successfully detected up to 20 antigens on a single slide. Combining image analysis and artificial intelligence software, for example with HALO (Indica Labs), the assay assembles high-dimensional images and enables quantitative histology and single-cell spatial analysis. Using this advanced method, we were able to identify rare cell populations, such as double-negative T cells, that are challenging to detect conventionally. ConclusionWe have developed a validated, high-capacity sIHC protocol that uses standard IHC procedures with commercially available, clinically validated off-the-shelf antibodies. This method is a valuable, cost-effective tool for obtaining extensive, high-dimensional single-cell-resolved spatial data from limited pathology samples, such as a human kidney biopsy.

Uniform, reproducible blood smears are critical for reliable hematological evaluation. Manual smear preparation, however, is user-dependent and introduces variability that limits quantitative microscopy. Here we developed BUsmear, a low-cost, 3D-printed, motorized blood smear device that prepares two smears simultaneously from a printed stage and a micro-motor drive with tunable linear velocity, controlled through a joystick-operated driver module. By spreading two slides in parallel with fully repeatable slide-to-slide motion, the device doubles throughput while eliminating operator-dependent motion artifacts, and can be fabricated on any benchtop 3D printer in under one day. To validate smear quality, we analyzed blood films from three donors together with a manual smear prepared by an expert from the blood of one of the same donors, giving a matched device-versus-manual pair. Automated Cellpose segmentation of 14,046 red blood cells across 12 bright-field fields showed that cell diameter was preserved and closely matched the expert smear (6.7-7.8 um across groups, within the 6.2-8.2 um human reference range; 6.7 vs 6.9 um in the matched pair), and that all films formed non-aggregated monolayers (Clark-Evans index of aggregation 1.04-1.24). Critically, red blood cells in the device films were markedly more circular than in the expert manual smear (mean eccentricity 0.405 vs 0.502; 0.443 vs 0.502 in the matched pair), with complete separation between the two methods at the level of whole fields of view. Because eccentricity reports smear-induced cell distortion, this indicates that a constant, mechanically controlled spreading velocity preserves red blood cell morphology better than skilled manual technique. BUsmear offers an accessible route to standardized smear geometry for quantitative analysis, including AI-based morphometry, and its low cost may be particularly advantageous in low-income countries with a high prevalence of malaria.

CD8 T cells play a central role in immune responses to infection and cancer. However, the diversity of T cell receptor (TCR) specificities makes it challenging to study the mechanisms that regulate T cell activation, differentiation, and effector function. Beyond TCR transgenic mouse models, various complex genome-editing approaches have been employed to overcome this challenge. However, these strategies are often technically demanding, time-intensive, and difficult to adapt. Investigators who are interested in testing de novo TCRs under their chosen experimental conditions would benefit from a standardized and accessible method. Here, we describe a protocol that combines ribonucleoprotein (RNP)-based CRISPR-Cas9 editing with retroviral transduction to enable efficient genetic manipulation of murine CD8 T cells. We show that T cells engineered via this protocol can be generated at sufficient scale for downstream in vitro assays and in vivo adoptive transfer experiments. We expect this method will be useful for investigators who require a standardized and accessible way to study how TCR specificity impacts CD8 T cell responses.

T cell repertoire sequencing has unveiled a vast, complex array of T cells responsible for the human immune responses. Traditional analytic methodology fails to fully characterize and quantify the diversity of T cell receptors constituting the T cell repertoire. T cell receptor clonal frequency measured in terms of T cell receptor beta (TRB) V gene segment usage when arrayed in correspondence with the respective V gene segment positions on the TRB loci yields a periodic, undulating curve in the spatial domain of the TRB genomic locus. Using the genomic distance from the TRB-D1 segment to the TRB-V1-29 segments, Fourier analysis was performed utilizing Lomb-Scargle periodogram to obtain Spectral Power curves quantifying the TRB V clonal frequencies from 6 allogeneic stem cell transplant donors (baseline) and recipients (>/=100 days) using a variety of analytic software. Spectral Power curves revealed dominant spectral peaks at wavelengths ranging from 4-9 kb (113-252 millicycles/kb) in the six donors, with consistent frequency domain spectral patterns. This is consistent with similar use of V segments across healthy individuals. Recipients on the other hand demonstrated more dispersed spectra, with a spectral centroid shifted towards higher frequencies compared to donors (260 vs. 247 millicycles/kb). Consistent with this observation, the Low Frequency Index was lower in the recipients (0.18 vs 0.20). Power was concentrated in the <3 kb and 3-12 kb wavelengths in both groups. The analyses reported here demonstrate that the healthy SCT donors have a remarkably similar spectral signature occupying short to intermediate wavelegnths in the frequency domain, whereas recipients tend to shift towards higher frequencies. These findings are consistent with a normal organized distribution of TRB V segment usage in healthy individuals (by analogy other loci), and a more diffuse and disorderly usage in recipients, consistent with the notion of T cell responses constituting a dynamical system which evolves as a function of time. Fourier analysis of TRB (and potentially TRA) sequencing data provides a repertoire wide summary of T cell clonal distribution.

We describe here an efficient method for gene editing in mouse T cells, based on well-established, high-efficiency retroviral transduction protocols. Our platform allows analysis of mutant phenotypes in primary murine T cells in vitro and in vivo. This approach uses a single retroviral vector to simultaneously knockout an endogenous gene and ectopically express its mutant version. This knockout/re-expression vector can be used as the only plasmid to transduce Cas9-expressing T cells, or used together with a Cas9 retroviral vector to transduce T cells from any mouse strain. We validated the system for analysis of murine T cells by targeting key molecules in proximal T cell signaling, i.e. CD3{gamma} and Zap70. We obtain high knockout and re-expression efficiencies in both Cas9-expressing and non-Cas9 T cells. Knockout efficiencies can be further improved by gRNA multiplexing. Endogenous proteins compete with their ectopically expressed mutants or tagged versions for cellular location, protein interactions and cellular functions. Here, we quantified the incorporation of CD3{gamma}-GFP into surface T cell receptor (TCR) complexes. Our data shows that the knockout and re-expression platform improves integration of CD3{gamma}-GFP into the TCR. Therefore, eliminating competition between endogenous and ectopic proteins benefits analyses of protein assemblies and signaling pathways in primary T cells. Furthermore, we validated advantages of our system for mutant analysis using wild-type and mutant Zap70s. Zap70 mutants deficient in TCR binding or kinase activity show their phenotypes only in the absence of endogenous protein, further validating our knockout/re-expression approach. Most importantly, this system can be used to generate gene-edited primary T cells for in vivo studies, such as the quantification of anti-tumor responses. Our knockout and re-expression platform provides a useful gene editing tool for primary T cells in fundamental research and immunotherapy development.

Nile Red is widely used for the detection of microplastics because its fluorescence emission is sensitive to local polarity and can distinguish hydrophobic plastics from hydrophilic ones. The fluorescence of the molecular rotor, 9-(dicyanovinyl)-julolidine (DCVJ) is less sensitive to polarity but more to viscosity. DCVJ is less widely used for microplastic analysis, although it has been used to detect polystyrene nanobeads. Here, we compared these dyes with standard samples from the Hawaii Pacific University Polymer Kit 1.0 and confirmed that Nile Red, in general, was better for the detection and identification of microplastics. Fluorescence emission was analyzed using photography, as well as spectroscopy. The color and peak emission wavelength of some stained environmental microplastics were affected by additives. Raman spectroscopy was used to confirm the chemical identity of such samples. Although DCVJ emits green fluorescence on binding to some microplastics, a peak at 620 nm has been reported with polystyrene nanobeads, attributed to dimer/excimer formation. We confirmed this property and directly observed diffraction-limited spots using fluorescence microscopy, attributed to single or just a few nanobeads. Nile Red also stains polystyrene nanobeads and gave stronger signals than with DCVJ, but Nile Red was prone to false positives due to dye aggregation in aqueous solutions.

Advances in immunophenotyping of tumour-infiltrating lymphocytes (TILs) have improved our understanding of prognostic biomarkers and immune targets in human melanoma. However, whether the tumour-immune landscape in canine oral malignant melanoma (COMM) is concordant with that of human melanoma has not been properly defined. To address this gap, we developed a 16-colour spectral flow cytometry panel to characterise TIL phenotypic and functional profiles in COMM. Validation using mitogen-stimulated peripheral blood mononuclear cells from healthy dogs (n = 5) demonstrated robust identification of major T cell lineages, including regulatory T cells (Tregs) and memory subsets, and reliable evaluation of their activation and exhaustion status. COMM patients (n = 8) displayed a distinct protumour microenvironment, characterised by an increased proportion of Tregs, enrichment of tumour-specific exhausted-like T cells co-expressing programmed cell death protein 1 (PD-1) and tumour necrosis factor receptor 2 (TNFR2), and a reduction in cytotoxic CD8 and natural killer T (NKT) cell populations compared with tissue-resident (n = 4) and circulating (n = 8) lymphocytes. Analysis of additional solid tumours, including a mast cell tumour, nerve sheath tumour and adrenal cortical carcinoma, further supported the capability of the panel to identify similar patterns of immune dysregulation across diverse canine tumour landscapes. Collectively, this work describes the first detailed evaluation of canine TIL immunophenotyping using spectral flow cytometry and provides insights into the immunosuppressive mechanisms shaping the tumour microenvironment in COMM. These findings not only increase our understanding of canine tumour immunology but also identify potential immune targets and support ongoing comparative immuno-oncology efforts.

Objectives: Capturing adequate blood volume for blood cultures is critical for accurate detection of bloodstream infections. Pediatric volume targets vary by age and weight, whereas adult targets are standardized. The BD BACTEC FXI Culture System (FXI) contains an integrated calibrated load cell capable of automatically reporting blood volume measurements for each vial loaded onto the system. This study evaluated the accuracy of the FXI's blood volume measurements in simulated pediatric and adult patients. Methods: Mock pediatric and adult blood draws were performed, using bagged whole blood, to replicate real-world collection protocols. Syringe-collected blood volumes ranged from 2.0 to 15.0 mL for pediatric patients, depending on mock patient weight, and were fixed at 40.0 mL for adults. Samples were inoculated into BD BACTEC Peds Plus/F, Plus Aerobic/F, and Lytic/10 Anaerobic/F Culture Vials, with a target volume of 2.0 to 10.0 mL per bottle. Reference blood volumes were determined gravimetrically using manually obtained pre- and post-inoculation weights with a blood-specific gravity of 1.055 g/mL and were compared to the automatically measured, gravimetric-based blood volumes reported by the BACTEC FXI Culture System. Results: Automated volume estimates were accurate to a mean error of -0.03 mL per bottle (SD, 0.40 mL; n=168; 95% CI, -0.09 mL, 0.03 mL) and -0.08 mL (SD, 0.79 mL; n=72; 95% CI, -0.26 mL, 0.10 mL) when assessing total volume collected per patient. Conclusions: Our findings demonstrate that the automated system can quantify blood volumes in BACTEC culture vials and support blood volume monitoring for pediatric and adult collections. The gravimetric approach is also amenable to full automation for efficient and accurate blood volume determination.

Background: Chronic inflammation is the most common histological feature in Benign Prostatic Hyperplasia (BPH), and T cells are a key component of immune infiltrate. Advanced BPH is commonly associated with the formation of nodules, but it remains unclear whether a link exists among T cell infiltration, nodular development, and BPH progression. Using a Toxoplasma gondii (T. gondii) model and human specimens, we characterize the subtypes of T cells present during prostatic hyperplasia and their association with nodular development of the prostate. Methods: Male CBA/j mice were intraperitoneally infected with T. gondii parasites, and flow cytometry was performed on the prostate to quantify the number of CD4+ and CD8+ T cells. Histology was used to score microglandular hyperplasia (MGH), and immunofluorescence was used to quantify and examine the locality of CD4+ and CD8+ T cells and compared that to human BPH tissue. Results: We found that infecting male mice with T. gondii resulted in an increase of both CD4+ and CD8+ T cells in the prostate acutely and that CD8+ cells remained sustained at chronically. We also established the presence of glandular nodule formation at this timepoint through hematoxylin and eosin (H&E) staining. Immunofluorescence revealed that CD8+ cells were found proximal to forming glandular nodules relative to non-nodular glands. We also found more CD8+ cells localized to non-nodular glands in nodular BPH tissue versus non-nodular BPH tissue. Finally, we discovered a higher prevalence of CD8+ cells in T. gondii IgG+ patients than in IgG- patients. All T. gondii IgG+ patients exhibited nodular BPH, whereas all but one IgG- patient exhibited non-nodular BPH. Conclusions: This study is the first to investigate the presence and location of CD4+ and CD8+ T cells within nodular and non-nodular BPH glands. We found an association of the presence of CD8+ T cells with nodular progression. This association held true in human prostate tissue. Translationally, CD8+ T cells may enhance nodular BPH progression, and T. gondii infection may promote this CD8+ T cell-mediated response.

Introduction: C3 glomerulopathy (C3G) is driven by fluid-phase alternative complement pathway dysregulation, with emerging evidence linking glomerular neutrophil infiltration to disease severity. Neutrophil extracellular traps (NETs) are implicated in other forms of glomerulonephritis. However, their participation in the pathogenesis of C3G remains undefined. Methods: Kidney biopsies from 33 patients with C3G (15 with dense deposit disease [DDD] and 18 with C3 glomerulonephritis [C3GN]) were compared with 15 anti-neutrophil cytoplasmic antibody associated vasculitis (AAV) biopsies as a neutrophil-rich disease control in this retrospective cross-sectional study. Glomerular neutrophils and NETs were identified using immunofluorescence, staining for myeloperoxidase, citrullinated histone H3, peptidyl arginine deiminase-4, and DNA. Supervised machine learning was used to quantify glomerular NET formation, and the data were correlated with kidney function at time of biopsy using linear regression. Results: Intraglomerular NETs were abundant and detected in the majority of glomeruli in C3G biopsies. Compared with AAV, C3G showed a significantly higher fraction of neutrophils forming NETs, despite similar neutrophil counts per glomerulus. NET abundance was similar in DDD and C3GN. In exploratory analyses, a greater proportion of glomeruli containing NETs was associated with lower kidney function (estimated glomerular filtration rate) at biopsy, and this association remained significant after adjustment for age, C3G subtype, and interstitial fibrosis. Conclusions: These observations demonstrate that intraglomerular NETs are a common and prominent observation in C3G and are associated with reduced kidney function at biopsy. These findings raise the possibility that NET deposition in glomeruli is a previously unrecognized driver of glomerular injury in C3G.

Hybridoma screening in semi-solid medium typically employs antigens labeled with visible fluorophores (e.g., FITC, AF488) to enable single-step identification of antibody-secreting clones. However, conventional chemical conjugation via NHS-esters or isothiocyanate groups frequently modifies lysine residues located within epitopes, potentially abrogating antibody recognition of these critical regions. Here, we describe a SpyTag SpyCatcher-based site-specific labeling strategy that circumvents epitope damage during semi-solid medium screening. A 16-amino-acid SpyTag was genetically fused to the C-terminus of the target antigen, enabling covalent conjugation to an sfGFP SpyCatcher fluorescent probe. In semi-solid medium supplemented with SpyTag-antigen and sfGFPSpyCatcher, positive hybridoma clones were readily identified by distinct fluorescent halos, whereas negative clones showed no detectable signal. Notably, the site-specific method yielded a significantly higher frequency of fluorescence-positive clones compared to the conventional AF488-labeled antigen method, suggesting that epitope preservation enhances screening recovery. Furthermore, this approach did not impair hybridoma growth or final clone positivity, offering a simple, rapid, and epitope-compatible method for monoclonal antibody screening.

Background: Tumor necrosis factor- inhibitors such as infliximab and adalimumab have transformed autoimmune disease treatment; however, infliximab is a mouse-human chimeric antibody that remains immunogenic, is associated with self-association/aggregation liability, and requires prolonged intravenous administration. We humanized infliximab and engineered infliximab-derived candidates with improved potency and developability. Methods: Infliximab complementarity-determining regions were grafted onto human germline frameworks to generate humanized infliximab. STage-Enhanced Maturation (STEM) technology produced an affinity-matured clone (hInBG4), followed by targeted amino-acid substitutions in the complementarity-determining regions to generate LW2Y, LW2YR2S, and LW2YHR1K. Variants were evaluated by a cell-based tumor necrosis factor alpha neutralization assay, affinity-capture self-interaction nanoparticle spectroscopy, a baculovirus particle enzyme-linked immunosorbent assay, size-exclusion high-performance liquid chromatography, transient expression in human embryonic kidney 293 cells, and tumor necrosis factor alpha binding kinetics by biolayer interferometry, including dissociation at pH 7.4 and 5.8. Results: All three variants showed two- to three-fold higher neutralization potency than chimeric infliximab and outperformed adalimumab. Affinity-capture self-interaction nanoparticle spectroscopy shifts decreased from double-digit parental values to low single digits, while baculovirus particle binding ratios remained acceptable. Size-exclusion chromatography showed cleaner monomer peaks with reduced tailing, and expression increased relative to humanized infliximab. LW2Y combined very high affinity at pH 7.4 with markedly faster dissociation at pH 5.8, consistent with pH-dependent antigen release. Conclusions: Humanization, affinity maturation, and targeted complementarity-determining region re-engineering generated infliximab-derived candidates with improved potency and developability and identified LW2Y as a lead for further preclinical evaluation.

Bone marrow chimeras are widely used to study immune development and function. As the field moves from cesium-137 (137Cs)-based irradiators to X-ray irradiators for safety reasons, it is essential to determine if there are differences in immune system reconstitution after irradiating mice with one of these two radiation sources. Here, we performed a comprehensive immunological comparison of mice lethally irradiated with 137Cs or one of two different X-ray platforms and reconstituted with congenic bone marrow. Mice received 12 Gy total body radiation in two 6 Gy sessions followed by intravenous transfer of donor hematopoietic stem cells and were analyzed eight weeks post-transplant. We assessed mouse survival, donor chimerism, immune cell subset distribution, and activation states across multiple organs (bone marrow, spleen, lymph nodes, liver, and lung). All groups exhibited comparable survival and high levels of donor chimerism, with expected organ-specific reconstitution patterns. Immune lineage distributions, CD4/CD8 ratios, and activation states did not differ by irradiation type. Host-derived radioresistant cells were also similar across all irradiation groups and were predominantly composed of T cells skewed toward an activated phenotype. Overall, our data show that X-ray irradiation with proper filters and energy levels (225 KVp and 320 KVp) can yield equivalent immunological outcomes, including immune reconstitution and activation states, as compared to the same radiation dose from 137Cs-based irradiation in bone marrow chimera models. These results support the continued adoption of X-ray irradiation systems in place of 137Cs for generating bone marrow chimeras to be used across a wide range of immunologic studies.

Antibodies exhibit species-specific sequence and structural features that influence their antigen-recognition properties. Although several studies have investigated porcine antibodies, their repertoire and structural characteristics remain less well characterized than those of several other mammalian species. In this study, we analyzed public porcine heavy-chain repertoire sequencing data together with available antibody structural data to identify characteristic features of porcine antibodies. We found several residues enriched in porcine antibody framework regions, particularly at the base of heavy-chain complementarity-determining region 3 (CDR-H3). In particular, Arg101 and Glu123 were closely positioned in available structures and may influence CDR-H3 conformation at its base, whereas Pro120 may help constrain local backbone conformation. We also observed non-canonical cysteine usage in both framework region 1 and CDR-H3, which may contribute to structural diversity in the porcine repertoire. Finally, we evaluated the humanization potential of a porcine antibody using a human antibody language model and found that human-likeness increased after model-guided substitutions, although the resulting sequences did not exceed the T20 score threshold. Overall, these results indicate that porcine antibodies possess distinct sequence and structural features that may influence CDR-H3 properties and should be considered in future antibody analysis and engineering.

The Bacillus cereus group comprises bacteria of biotechnological interest, but also raises health concerns. Some bacteria in this group are opportunistic human pathogens, mainly causing foodborne gastrointestinal infections. As of today, the presence, sequence variability, or expression of genes encoding toxins or other virulence factors are insufficient to predict the potential of a given isolate to cause the diarrheal form of the disease. To address this limitation, we developed a sandwich ELISA to quantify the NheA and Sphingomyelinase (SMase) proteins in culture supernatants to test them as markers of pathogenic potential. Application of the assay to a collection of B. cereus group isolates revealed that strains associated with food poisoning outbreaks produce significantly more NheA and SMase than those isolated from the environment or from commercial products. Statistical analyses show that the combined quantification of NheA and SMase provides robust discrimination between pathogenic and non-pathogenic (environmental and commercial) profiles. These results demonstrate that the quantitative assessment of both NheA and SMase production can serve as a reliable biomarker for distinguishing diarrheic food poisoning isolates from harmless strains.

Simultaneous quantification of monoglycosphingolipid stereoisomers is required to monitor changes in defective enzymatic pathways linked to diseases such as Gaucher Disease, Parkinson's Disease, and Krabbe Disease. Resolution of beta-glucosyl and beta-galactosyl epimers cannot be achieved by standard liquid chromatography, electrospray ionization, tandem mass spectrometry (LC-ESI-MS/MS). Separation becomes possible when field asymmetric ion mobility spectrometry (FAIMS), also known as differential mobility mass spectrometry (DMS), is added as an orthogonal separation technique to LC. FAIMS/DMS separates epimeric ion clusters in a high versus low electric field (separation voltage, SV) then redirects the target epimeric ions to the mass spectrometer through the application of a direct current (compensation voltage, CoV). Resolving SVs and CoVs must be manually determined for each lipid. Manual derivation is a labour-intensive process that requires pure synthetic standards, limiting the number of stereoisomers a user can include in an assay. To address this problem, we introduce here intelligent DMS (iDMS). iDMS is an in silico supervised neural network model that learns the ion mobility relationships between SV and CoV and the monoglycosphingolipid structural features of sugar headgroup, N-acyl chain length, and N-acyl degree of unsaturation. iDMS predicts the SV and CoV combinations capable of resolving any stereoisomer pair from a training dataset of composed of measured signal intensities across a range of SVs and CoVs of 12 lipids. This machine learning alternative to manual DMS optimization promises to accelerate the deployment of multiple-reaction-monitoring mode (MRM) RPLC-ESI-DMS-MS/MS assays for the routine and rapid quantification of biologically relevant monoglycosphingolipid stereoisomers.

Since model dose-response is critical when assessing caries lesion development over time, this study evaluated the influence of fluoride dose and treatment duration on caries progression in a rat caries model. Streptococcus mutans-infected Sprague-Dawley rats were treated with deionized water, 226 ppm F-, or 2,260 ppm F- twice daily for 3, 4, or 5 weeks. Caries lesions were assessed using Larson's modification of the Keyes scoring system and complemented by micro-computed tomography (microCT). Intraoral fluoride availability, serum and bone fluoride concentrations and microbial counts were also determined. Fluoride reduced caries severity in a dose- and time-dependent manner. While early enamel lesions were detected in all groups, extensive dentine lesions increased over time, in a dose-dependent manner, in the control and 226 ppm F- groups, and were not observed in the 2,260 ppm F- group after 5 weeks. Intraoral and bone fluoride availability increased significantly with fluoride concentration and treatment duration, whereas serum fluoride levels reflected fluoride dose instead of treatment duration. MicroCT-derived enamel volume correlated negatively with both total and extensive caries scores, supporting its utility as an objective measure of lesion severity. In conclusion, extending model length from 3 to 5 weeks increased the severity of caries lesions in a dose-dependent manner. Fluoride intraoral availability and bone fluoride also demonstrated a dose and time-dependent response.

Background: Late-onset neutropenia (LON) is an infrequently reported, unpredictable side effect of anti-CD20 therapy, with incidence varying by agent, diagnosis, and screening protocol. Objective: The primary objective of this cross-sectional, retrospective study was to estimate the proportion of patients who developed LON over 13 months (April 2023-April 2024). Methods: Consecutive adult patients diagnosed with central nervous system (CNS) autoimmunity who received at least one rituximab(RTX) or ocrelizumab(OCR) infusion between January 2016 and March 2024 were included; patients who switched to another immunotherapy, had no post-treatment blood draw, or had unverifiable infusion records were excluded. LON events were assessed using all post-treatment CBCD blood draws during this period. Results: A total of 171 patients were enrolled: 141 received rituximab and 30 received ocrelizumab. A total of 319 post-treatment blood tests were performed. Sixteen patients (16/171) had neutropenia (9.4%, 95% CI 5.8-14.7): 12 on rituximab (8.5%) and 4 on ocrelizumab (13.3%; p=0.487). LON occurred at a median of 158 days (130-188) since the last infusion. All patients were asymptomatic, mostly had Grade 1 neutropenia (15/16, 93.8%). BMI (22.2 vs. 27.5 kg/m2, p=0.001) and prior natalizumab exposure (37.5% vs. 14.2%, p=0.023) were significantly different between neutropenic and non-neutropenic patients. Conclusion: The proportion of patients with LON in this cohort was higher than most previously reported, with all cases asymptomatic. Lower BMI and prior natalizumab exposure emerged as potential risk factors warranting further investigation. Larger, prospective studies with standardized surveillance are needed to establish the true frequency and risk factors.

BACKGROUND: The transport and storage conditions of thawed plasma are not strictly regulated by the FDA and applying red blood cell transport standard of 1-10 degrees Celcius to recently thawed plasma often results in high discard rates. This study evaluated the extended 120-hour (5-day) coagulation factor stability and sterility of thawed plasma frozen within 24 hours (PF24) following a 6-hour transport cooler simulation. STUDY DESIGN AND METHODS: Fourteen PF24 units (8 group O, 6 group B) were thawed at 30-37 degrees Celcius and assigned as control (n=7, direct 1-6 degrees Celcius refrigeration) or experiment (n=7) units. Experiment units were held at room temperature for 30 minutes, stored in validated transport coolers for 6 hours, and then transferred to 1-6 degrees Celcius refrigeration. Measurements of temperature, prothrombin time (PT), Factor V (FV) activity, and Factor VIII (FVIII) activity were conducted at 0-, 6-, 24-, and 120-hour post-thaw. Sterility testing was performed at 0-hour and 120-hour using automated aerobic and anaerobic blood cultures. RESULTS: No statistically significant differences were observed between control and experiment units at 120-hour for mean PT (15.09 vs. 15.16 seconds, p = .44), FV activity (81.14 vs. 74.57%, p = .23), or FVIII activity (61.86 vs. 53.00%, p = .22). Delta analysis (120h-0h) confirmed equivalent factor decay rates between groups. All bacterial cultures showed no growth at 120-hour. CONCLUSION: A 6-hour cooler time of thawed PF24 does not accelerate coagulation factor degradation or compromise sterility over an extended 5-day shelf life. These findings validate flexible inventory return policies, allowing blood banks to reduce product waste.

Click-qPCR is a browser-based application for relative qPCR analysis that requires a tidy-format CSV file containing four columns: sample, group, gene, and Cq. Preparing this input from qPCR instrument output typically requires manual reformatting and calculation of mean Cq values for technical replicates. To simplify this process, we developed Click-Prep (https://kubo-azu.shinyapps.io/Click-Prep/), an interactive web-based application designed specifically to create Click-qPCR input files. Click-Prep imports CSV, TXT, TSV, and XLS/XLSX files and supports skipping of instrument-generated metadata rows, interactive column mapping, and manual assignment of experimental groups. Users can review technical-replicate measurements, exclude selected rows according to predefined quality-control criteria, and calculate mean Cq values for each sample-group-target combination. Missing or nonnumeric Cq values are flagged for review and must be resolved before the mean is calculated. Click-Prep can also combine compatible formatted CSV files, such as datasets obtained from separate qPCR plates. The resulting dataset is exported as a standardized CSV file containing the four fields required by Click-qPCR. By integrating these operations into a guided browser-based workflow, Click-Prep enables users to prepare Click-qPCR input files rapidly and consistently without programming.