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Introduction Establishment of reference intervals (RI) for quantitative assays requires a minimum of 120 qualified normal donor (ND) samples for each partition of an analyte. Based on CLSI EP28 A3C guideline [CLSI. Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline-Third Edition. CLSI document EP28-A3c. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.], verification of previously established and/or published RI can be performed with a minimum of 20 NDs, especially given complexities of finding qualified NDs. Reagent manufacturers typically provide adult RI, thus facilitating RI verification as a viable option. NDs for the Pediatric (Peds) (<18 years of age) population are difficult to obtain, published RIs are performed with sample cohorts less than the suggested number or only performed with specific instrument/reagent combinations. Herein, we report our experiences with Peds RI. Methods After IRB approval, healthy pediatric outpatients ages 0-18yrs were recruited and consented. Plasma obtained from umbilical cord, finger/heel/ear stick, or venipuncture blood samples (no more than 50 mL or 3 mL per kg) and collected in 3.2% sodium citrate. Assays included Protein (P) C antigen (PCAG) and chromogenic activity (PCFX); P S total (PST), free (PSF) antigen, and functional (SFX); antithrombin antigen (ATAG) and chromogenic activity (ATFX); plasminogen (PLGN) and alpha-2-plasmin inhibitor (A2PI). Assay kits were PCFX - HemosIL® (Werfen, Beford, MA) Protein C, PST - LIATEST® (Diagnostica Stago S.A.S., Asnières sur Seine, France) Protein S, PSF - HemosIL® Free Protein S, SFX - HemosIL® Protein S Activity, ATAG - LIATEST® AT III , ATFX - HemosIL® Liquid Antithrombin, PLGN - HemosIL® Plasminogen, and A2PI - HemosIL® Plasmin Inhibitor performed on the ACL TOP 700 (Werfen) instrument. PCAG - REAADS® Protein C Antigen Test Kit (Corgenix, Inc. Broomfield, CO) was performed on the Janus G3 liquid handler (Revvity, Waltham, MA) and Synergy Neo2 microplate reader (Agilent, Santa Clara, CA). Assay mean and range were calculated. Results Insufficient numbers of patients <1yr were collected. Type of assay, age range in yr and number of samples (n), assay mean in % and range results respectively were as follows: PCAG 1-5 (n=15) 87(46-146); 5-10 (n=82) 81 (45-158); 10-15 (n=169) 84 (44-157); 15-18 (n=107) 91 (57-149). PCFX 1-5 (n=20) 97 (56-140); 5-10 (n=84) 89 (62-135); 10-15 (n=188) 91 (55-140); 15-18 (n=124) 96 (54-138). PST (males): 1-5 (n=10) 120 (96-145); 5-10 (n=46) 108 (75-150); 10-15 (n=91) 111 (80-168); 15-18 (n=63) 113 (72-184). PST (females): 1-5 (n=9) 104 (67-146); 5-10 (n=37) 111 (81-159); Ages 10-15 (n=94) 107 (78-153); 15-18 (n=63) 101 (65-148). PSF (males): 1-5 (n=10) 94 (74-109); 5-10 (n=47) 101 (66-141); 10-15 (n=93) 101 (69-151); 15-18 (n=62) 111 (74-187). PSF (females): 1-5 (n=9) 78 (9-124); 5-10 (n=37) 107 (79-137); 10-15 (n=95) 99 (63-138); 15-18 (n=63) 97 (60-132). SFX (males): 1-5 (n=9) 100 (85-128); 5-10 (n=48) 104 (66-144); 10-15 (n=93) 103 (66-168); 15-18 (n=59) 109 (72-157). SFX (females): 1-5 (n=8) 94 (18-121); 5-10 (n=37) 107 (81-143); 10-15 (n=90) 101 (65-147); 15-18 (n=60) 97 (53-135). ATAG 1-5 (n=19) 104 (80-121); 5-10 (n=83) 104 (78-121); 10-15 (n=188) 104 (66-126); 15-18 (n=126) 101 (67-126). ATFX 1-5 (n=20) 112 (94-135); 5-10 (n=84) 107 (87-123); 10-15 (n=188) 105 (84-138); 15-18 (n=126) 101 (84-129). PLGN 1-5 (n=17) 96 (76-124); 5-10 (n=85) 88 (65-125); 10-15 (n=183) 90 (49-151); 15-18 (n=119) 94 (64-151). A2PI: 1-5 (n=17) 134 (118-151); 5-10 (n=85) 126 (107-139); 10-15 (n=183) 121 (104-140); 15-18 (n=118) 116 (91-143). Conclusions Based on CLSI EP28 A3C criteria for verification, no more than 10% of results should fall outside of established/published RI, for example adult RI. Thus, compared to the SCL adult RI, PLGN assay meets criteria in all age ranges and no additional ND are needed. For the remaining assays, additional ND for ages 1-5 are needed. For ages 5-18 criteria for verification are satisfied for all analytes except PCAG. PCAG has 27, 61, and 19 patients falling below lower RI for ages 5-10, 10-15, and 15-18, respectively.

Trauma-induced coagulopathy (TIC) is common after injury and reliable quantification of its severity is lacking. The objective of this study was to evaluate whether thrombin generation would be significantly altered if whole blood samples from trauma patients were collected without (citrate-only) and with corn trypsin inhibitor (CTI+). In a prospective cohort study, 36 trauma pts presenting to a Level I Trauma Center within 12 hours of injury, had whole blood collected simultaneously in citrate-only (3.2%) and citrated plus CTI (CTI+ [final 25 ug/mL]), and processed to platelet free plasma by double centrifugation (3000g, 15 minutes). Samples were stored at -80C with no thawing until analysis. Thrombin generation profiles were measured using the ST Genesia assay (Diagnostica STAGO SAS) and parameters included endogenous thrombin potential (ETP), lag time (LT), peak height, start of tail, velocity index, and time to peak (ttPeak) were analyzed for trauma pts. Data presented as median [IQR] or n (%), with paired t-test and Spearman's correlation analysis were performed between citrated non-CTI and citrated CTI spiked blood samples, with p-value <0.05 statistically significant. Pts presented with significant injury ISS 13 [5, 21], age 45 [29, 59] years, 80.6% male, BMI 26.9 [25.4, 28.3], 94.4% blunt trauma, 22.3% had surgery by day 1 of admission. Significant differences were found between the citrated-only and CTI +tubes in several thrombin generation parameters: ETP (1339 Nm*min [1172.5,1417], 1124.5 [925.4,1285], p<0.001), peak height (222.3 Nm [186.0, 275.6], 160.6 [132.7,208.3], p<0.001), ttPeak (5.64 min [4.86,6.63], 6.19 [5.36, 7.34], p<0.001), start of tail (19.4 min [17.6,21.3], 21.6 [18.9, 23.6], p=0.014), and velocity index (117.6 Nm/min [77.9, 163.1], 71.9 [54.9,106.0], p<0.001). However, LT (3.14 min [2.74, 3.90], 3.08 [2.84, 3.96], p=0.665) did not show a statistically significant difference. Spearman's correlation analysis showed high correlation between citrate-only and CTI+ citrated plasma samples, with Spearman's rank correlation coefficients for LT (0.941), peak height (0.760), ttPeak (0.928), ETP (0.770), start of tail (0.869), and velocity index (0.766) respectively. The results suggest that use of CTI+ decrease thrombin generation kinetics from blood samples obtained from trauma patients. These findings underscore the need for careful consideration of sample collection and processing for use of thrombin generation assays in clinical setting. However, there is very strong correlation of the thrombin parameters obtained from citrated-only plasma to CTI+ plasma samples, suggesting they capture similar information. CTI-spiked citrated tubes appear to reduce contact activation with respect to thrombin generation profiles compared to standard citrated only tubes. This should be a consideration for platform-based lab processing of thrombin generation assays for assessment of coagulopathy after injury; however, given the cost of CTI, citrated-only tubes can be an option for characterization of thrombin generation profile in clinical samples.

ABSTRACTBackgroundCoccidioidomycosis is a fungal infection that poses a serious risk when transmitted through organ transplantation. We analyzed cases reported to the Organ Procurement and Transplantation Network ad hoc Disease Transmission Advisory Committee from 2013 to 2022.MethodsDonors and/or recipients who had positive Coccidioides immitis/posadasii serology, pathology, and/or culture were included in this study. Cases adjudicated as ‘proven’ or ‘probable’ were analyzed for donor infection risk factors, the timing of infection, transmission by organ type, clinical manifestations, and recipient outcomes. Patient and facility identifiers were removed prior to review.ResultsDuring this time period, 73 potential instances of Coccidioides donor disease transmission events were reported. Among them, infection was transmitted from seven deceased donors to eight recipients. All seven deceased donors had prior infection or exposure to regions where coccidioidomycosis is endemic. Of 20 individuals receiving organs from these donors, eight developed infection, resulting in a 40% transmission rate. The median time to diagnosis post‐transplant was 39 days. Disseminated disease occurred in six recipients, five of whom died from the infection. Notably, none of the recipients who received prophylactic antifungal treatment died from the infection.ConclusionDespite its rarity, donor‐derived Coccidioides infection is a serious concern, particularly due to the high mortality rate in the early post‐transplant period. To mitigate these risks, a thorough assessment of donor exposure history, coupled with donor serology and bronchoalveolar lavage cultures, can effectively guide post‐transplant antifungal prophylaxis. Prompt reporting is crucial to prevent Coccidioides infections among other recipients. image

Abstract The A1 domain in Von Willebrand Factor (VWF) initiates coagulation through binding to platelet glycoprotein GPIbα receptors. Von Willebrand Disease (VWD)-Mutations in A1 that either impair (type 2M) or enhance (type 2B) platelet adhesion to VWF can locally destabilize and even misfold the domain. We leveraged misfolding in the gain-of-function type 2B VWD phenotype as a target, distinct from the normal conformation. Two nuclease-resistant 2′-fluoropyrimidine RNA aptamers were selected to discriminate normal A1 domains from a type 2B V1314D A1 variant in a glycosylated A1A2A3 tri-domain VWF-fragment. Two aptamers, W9 and V1, were isolated that selectively recognize, bind, and inhibit the A1–GPIbα interaction with WT A1A2A3 and V1314D A1A2A3, respectively. These aptamers were tested against their respective recombinant targets, plasma VWF, VWF concentrates, and patient plasma with the heterozygous type 2B VWD R1306W variant using clinical assays, surface plasmon resonance and inhibition assays of platelet adhesion to recombinant A1 and A1A2A3 domains under shear stress. The specificity of W9 and V1 aptamers confirms that pathological conformations of VWD Type 2B proteins are different from normal VWF. The availability of aptamers that distinguish normal plasma-derived VWF from VWD suggests potential applicability in clinical diagnosis of severe gain-of-function phenotypes.

Abstract Background. Extracellular vesicles (EV) are emerging as new biomarkers for cancer diagnostics and therapeutic monitoring1. For example, in patients that receive PD1/PD-L1 immune checkpoint inhibitors (a key pillar of cancer immunotherapy), circulating EV-derived PD-L1 is gaining attention as a non-invasive biomarker for therapeutic efficacy. Standard methodologies to measure EV-derived PD-L1 requires ultracentrifugation to first separate EVs and then quantify those that express PD-L1 using nano flow cytometry. These are tedious processes that require trained technologists in centralized facilities, which is a key barrier to frequently monitoring EV-based biomarkers. Yet, frequent monitoring is crucial to quickly identify and stop an ineffective therapy while more fruitful options are still available. Method. To bridge the gap, we develop a proof-of-concept digital microfluidic (DMF) device that can separate EVs directly from biofluids and quantify PD-L1+ EVs automatedly. Briefly, DMF device supports automatedly droplet handling, including droplet transport, splitting and mixing, based on electrowetting principles2. Immunomagnetic beads were used to capture EVs from biofluids, and EVs were then detected by electrochemical sensors inserted into the device. These tips of the electrochemical sensors were modified with gold nanoparticle self-assembled layers to enhance target binding, and CD9 antibodies were immobilized on the tips to bind to EVs. We then used anti-PD-L1 as secondary antibodies for target detection. Differential Pulse Voltammetry (DPV)3 were performed to obtain the results. Results. We used the DMF device to extract 109- 1011 #/mL EVs secreted by a human breast cancer cell line (MDA-MB-231). Briefly, 10 mL of cell culture media was loaded into the DMF device, and the sample was mixed and incubated with a 10 mL droplet of immunomagnetic beads to capture EVs. After 5 min, EVs were eluted in 20 mL of elution buffer, and the beads were discarded. We estimated the number of EVs using Nanoparticle Tracking Analysis; the number of EVs extracted on-chip was comparable with those that were extracted using standard in-tube method. We also verified the purity of the EVs extracted on-chip using Western Blot. Our results showed that the electrochemical sensors can detect as low as 104 #/mL PD-L1+ EVs, with a linear range of 104 – 107 #/mL (R2=0.9701). The entire process can be completed within 1 hour. Conclusion. We integrated automated EV extraction and their surface marker detection in a single DMF device. Our next step is to extract and detect EV-based biomarkers from plasma samples. This platform holds promise for the regular monitoring of EV-based biomarkers that are indicators of therapeutic responses in patients that receive cancer immunotherapies. References1. F. Lucien, D. Gustafson, M. Lenassi et al, Journal of extracellular vesicles 12 (12), 12385 (2023). 2. Y. Zhang and Y. Liu, Sensors & Diagnostics (2022). 3. Y. Zhang, J. Liu, T.-W. Lo et al, Biosensors and Bioelectronics: X, 100490 (2024). Citation Format: Ting-Wen Lo, Yuqian Zhang, Jing Liu, Yohan Kim, Haidong Dong, Fabrice Lucien, Yuguang Liu. Rapid extraction and detection of extracellular vesicle-derived PD-L1 in a microfluidic platform [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A030.

Abstract Improved response assessment methods are needed for patients with gliomas. Intracranial cerebrospinal fluid (CSF) is longitudinally accessible and provides an abundance source of candidate biomarkers, such as cell-free DNA (cfDNA), for disease monitoring. To evaluate the clinical feasibility of molecular profiling from longitudinal intracranial CSF, CSF was acquired from patients with CSF access devices, including Ommaya reservoirs and ventriculoperitoneal shunts. cfDNA was extracted and analyzed via PredicineCARE (Next Generation sequencing) or PredicineSCORE (low pass whole genome sequencing). Five patients (2 females, 3 males; median age: 40 years, range 32-64 years) underwent longitudinal intracranial CSF collection via Ommaya reservoirs (n=4) or VP shunt (n=1). In total, forty-eight CSF samples were obtained (median volume: 4.00 mL; 0.5-5 mL), with 39 samples (81.3%) yielding sufficient cfDNA for variant profiling. Our initial findings suggest that tumor fraction increased by 6.28x, 2.87x, and 8.31x with radiographic progression in three patients. Tumor fraction decreased by 0.08x and 0.04x in two patients with paired immediate pre-versus-post chemoradiation samples. Despite ongoing pseudoprogression in one patient, tumor fraction decreased to unmeasurable levels from a post-resection baseline of 0.26. Patient-specific tumor-associated variant allelic frequencies (VAFs), including TP53, PTEN, TERT, and CDKN2A/B, decreased within individual patients after resection and chemoradiation. In two patients with isocitrate dehydrogenase (IDH) mutant gliomas, decreasing IDH1 VAF after resection and chemoradiation correlated with decreased CSF D-2-hydroxyglutarate (D-2-HG) levels (0.64x and 0.62x, respectively, for the first patient, and 0.01x and 0.07x for the other patient). Both CSF IDH1 VAF and CSF D-2-HG increased a patient who had radiographic progression (2.56x and 9.21x, respectively). Moreover, CNB decreased below the limit of quantification during treatment and increased above the limit at progression. In conclusion, longitudinal CSF cfDNA can feasibly be obtained via CSF access devices in patients with gliomas during their disease course toward evaluating candidate monitoring biomarkers.

Abstract BACKGROUND Glioblastoma (GBM), one of the deadliest primary tumors of the central nervous system, is characterized by a highly immunosuppressive tumor microenvironment (TME). Tumor-associated macrophages and microglia (TAMs) are a dominant population of immune cells in the GBM TME and substantially contribute to immunosuppression, tumor progression, and treatment resistance. Elucidating the molecular mechanisms underlying TAM behavior is crucial to the development of effective immuno-therapeutic strategies for treating GBM. The adaptor protein, Caspase Recruitment Domain-containing protein 9 (CARD9) is primarily expressed in myeloid cells and its activation triggers assembly of the CARD9-BCL10-MALT1 (CBM) complex, which then promotes activation of NF-κB and/or MAPK pathways. The aim of this study was to investigate the role of the CBM complex in the GBM TME and assess its potential as a therapeutic target. METHODS/RESULTS In silico analyses revealed that GBM tumors demonstrate increased CARD9 and MALT1 expression in comparison to non-tumor brain tissue, with CARD9 expressed only within the myeloid compartment. Using co-culture, scratch wound, and transwell assays, we found that GBM tumor cells induce macrophage M2 polarization and migration via a mechanism that is dependent on CARD9/MALT1 signaling within macrophages. scRNA-Seq and flow-cytometric analysis of orthotopic syngeneic immunocompetent GBM tumor models revealed that the GBM TME is dominated by macrophages. Disruption of MALT1 protease activity within the TME leads to reduced tumor infiltration by immunosuppressive TAMs, remodeling of TAMs toward an immunoreactive anti-tumor phenotype, and improved survival of GBM-bearing mice. Additionally, we found that tumor growth was abrogated when GBM-bearing mice were treated with either a MALT1-protease inhibitor or a CARD9 inhibitor. CONCLUSIONS Our study suggests that blockade of the CARD9-MALT1 signaling axis impairs GBM-induced macrophage recruitment and M2 polarization and inhibits tumor progression. These findings nominate the CARD9-MALT1 signaling axis as a new target for TAM-directed immunotherapy for the treatment of GBM.

Abstract Introduction: Immune checkpoint inhibitors (ICI) are an essential systemic therapy for advanced melanoma. However, most melanomas develop resistance to ICI. Tumor-derived soluble PD-L1 (sPD-L1) and other soluble immunosuppressive factors drive checkpoint inhibitor resistance and correlate with inferior survival. We previously showed that therapeutic plasma exchange (TPE) removes sPD-L1 from circulation. Thus, we hypothesized that TPE-mediated removal of sPD-L1 and other immunosuppressive factors could overcome immunotherapy resistance in refractory melanoma. Methods: In this clinical trial (NCT04581382), we prospectively enrolled eighteen (18) patients with widely metastatic melanoma with progression despite anti-PD-1 ICI and elevated sPD-L1 by ELISA (≥1.7ng/mL). Each patient received radiotherapy to between one and three metastatic lesions (at least two unirradiated lesions) followed by three TPE sessions on consecutive days and re-challenge with checkpoint inhibitor. The primary safety and efficacy endpoints of the study were adverse events (AEs) and sPD-L1 reduction by TPE, respectively. Secondary endpoints included RECIST-based response in unirradiated lesions and overall survival. Correlative studies included kinetics of sPD-L1 and soluble immonsuppressive factors and dynamics of peripheral immune cell phenotypes. Results: Mean age was 62 (SD 13) and seven of eighteen (39%) were female. Mean baseline sPD-L1 was 26.31 ng/mL (40.01). The treatment was well-tolerated with one (6%) patient with grade 3 and 4 adverse events from a central line infection after TPE. Levels of sPD-L1 were significantly reduced by TPE (mean 80.2% reduction, p<0.0001). Two patients (11.1%) experienced complete response (CR), one (5.6%) partial response, three (16.7%) stable disease, and 12 (66.7%) progressive disease. In one case, immunotherapy was discontinued after two years due to no detectable lesions. Changes in tumor-reactive (TTR) GZMB+/CX3CR1+/CD11ahigh and other peripheral immune cell populations predicted overall survival in this cohort. In addition to sPD-L1, other soluble mediators of ICI resistance were also reduced by TPE and predicted overall survival in this cohort. Summary/Conclusion: sPD-L1 and other soluble immunoregulatory signaling molecules are important mediators of ICI resistance. SBRT and TPE can resensitize ICI-refractory melanoma by removing these factors. Patients with persistently elevated or rapid rebound of sPD-L1 following TPE experienced poor response and overall survival. Serial monitoring of sPD-L1 may predict response to ICI and multiple courses of TPE may be necessary. Our findings may apply in other ICI-resistant cancers with elevated sPD-L1. ClinicalTrials.gov registration: NCT04581382, ReCIPE-M1 (Rescuing Cancer Immunotherapy with Plasma Exchange in Melanoma 1).