The influence of a new haemostatic material on surgical bleeding was evaluated in 100 patients who were prospectively randomized to either normal surgical gauze or calcium alginate swabs used throughout cholecystectomy (n = 40), simple mastectomy (n = 18) or inguinal hernia repair (n = 42). Overall, median (range) blood loss was 91 (3-329) ml for gauze and was significantly reduced by calcium alginate swabs to 72 (2-181) ml (P less than 0.05). Unexpectedly, operation times were also shortened from 45 (17-95) min for gauze to 35 (13-70) min with calcium alginate swabs (P less than 0.02). This reduction in blood loss and operating time was greatest for mastectomy, was still statistically significant for cholecystectomy, but was unimportant in inguinal hernia repair. Calcium alginate haemostatic swabs may become routine in major surgery, particularly where blood loss leads to the need for transfusion.

Calcium alginate (Kaltostat, Cair Ltd) is a new absorbable material for topical haemostasis in surgery. The possible mode of action, release of calcium ions in exchange for sodium was investigated in human blood.Calcium release was measured in 15 mg samples of calcium alginate placed in 20 ml of 0.9% saline, for intervals of 1, 3 or 10 minutes. To assess the effect on platelets, 3 mg of calcium alginate or surgical gauze were added to 5 ml of Heparinised (100 units) fresh blood for 2 minutes and platelet counts then made using plain blood as a control. Finally using a thrombelastograph, the activation of whole blood coagulation was assessed after a 2 minute contact with 3 mg of calcium alginate, surgical gauze or no additive as control.When calcium alginate was placed in saline, 26% of calcium ions were released in 1 minute giving a calcium ion concentration of 4.62 t 0.02 mmol/L, with only slight further release after 10 minutes to 4.82 ± 0.004 mmol/L. There was a corresponding decrease in sodium ion concentration. Adding calcium alginate to whole blood reduced the platelet count from a control value of 248 i 16 × 109/L to 222 f 15 × 109/L (p< 0.05) compared to 241 ± 15 × 109/L for surgical gauze. Similarly calcium alginate shortened whole blood coagulation time from 17-7 i 1.0 minutes control, to 12.9 ± 1-32 mins (p< 0.001) compared to 15.0 ± 1.5 mins (p< 0.02) for surgical gauze.Calcium alginate rapidly releases calcium ions in exchange for sodium on contact with blood stimulating both platelet activation and whole blood coagulation, significantly more than simple contact activation by surgical gauze.

Immunotherapy has demonstrated potential to reactivate or transfer T cell immunity and regress tumors, offering hope to relapsed or refractory diffuse large B-cell lymphoma (DLBCL) patients. However, many DLBCL patients do not experience therapeutic benefit, likely owing to a lack of pre-existing anti-tumor immunity and/or poorly understood immunosuppressive mechanisms in the tumor microenvironment (TME). Understanding the different obstacles that cytotoxic T cells face in the DLBCL TME will help the development of novel therapeutic approaches to overcome them and optimize immunotherapy. Stroma-associated gene signatures reflecting fibroblast and immune cell activity as well as angiogenesis have been associated with outcome in DLBCL but the biology underlying these signatures has been understudied. Here we have examined beyond tumor 'effacement' and hypothesized that, rather than being sparse bystanders, lymph node stromal cells may be important players in driving immune suppression in lymphoma. Multiplex immunofluorescence (IF) confocal microscopy analysis of the major stromal cell subsets revealed a marked expansion and remodeling of podoplanin, PDPN+ fibroblastic reticular cells (FRCs) in DLBCL lymph node tissue TME biopsies across GCB and ABC subtypes (n=40) compared to reactive control tissues (n=10). FRC myofibroblasts were similarly remodeled in tumors from the transgenic mouse model of DLBCL (Iμ-HABCL6, n=10) compared to wild type littermates (n=5). These altered PDPNhigh, αSMAhigh FRC networks were interspersed within effaced lymph node tissues in close proximity to DLBCL tumor cells. To model the interactions between tumor cells and FRCs, we established 2D and 3D co-culture platforms that combined DLBCL cells (or non-malignant control B-cells) and PDPN+ FRCs derived from human or murine lymph node tissues. These quantitative assays have shown that tumor cells activate FRCs promoting their proliferation, increased expression of PDPN, marked elongation/stretching and subsequent reduced ability to contract 3D collagen matrix (non-contractile) (P<.01). Exposure to tumor cells caused uncoupling of PDPN from RHOA signaling and redistribution of PDPN into lipid rafts, permitting FRC stretching. FRCs purified from both human and murine tumor lymph nodes showed the same activated morphology and phenotype, demonstrating that our co-culture systems recapitulate in vivo findings. Screening experiments have shown that lymphoma-expressed membrane and soluble lymphotoxins (LTα1β2, LTα3) as well as TNFα significantly contribute to the remodeling of FRCs (P<.01). Co-culture assays have revealed evidence for mutualistic interaction as FRCs, that express BAFF, promote the survival of DLBCL cells in 3D matrix gels (P<.01). Expanded lymphoma PDPNhigh FRCs in situ co-expressed BAFF compared to a more restricted expression profile (marginal zone FRCs) in reactive lymph node tissues. Flow cytometry revealed that lymphoma FRCs exhibit a cancer-associated fibroblast (CAF)-like immunophenotype including upregulation of fibroblast activation protein (FAP) and αSMA, as well as immunomodulatory MHC class I, PD-L1 and PD-L2 molecules compared to control FRCs (P<.01). An important function of FRCs in regulating immunity is attracting and maintaining T cells by secreting chemokines and promoting their migration along the network. Functional assays revealed that T cells show significantly reduced chemotaxis as well motility (quantitative time-lapse movies) across 2D and 3D lymphoma FRC networks compared to control FRCs (P<.01). Multiplex IF analysis revealed reduced CCL19 and CCL21-expressing FRCs in DLBCL compared to the reactive control FRC network as well as low numbers of tumor-infiltrated CD8+ T cells, which localized closely with remodeled FRCs. We next determined whether lymphoma FRCs (CAFs) could negatively regulate T cell function. Triple culture autologous assays (murine and human) have shown that prior exposure of tumor-infiltrated CD8+ T cells to FRCs significantly decreased their cytolytic killing activity against tumor cells (P<.01). In conclusion our data indicate that DLBCL tumor cells convert FRCs into immunosuppressive CAFs, which exhibit altered immumomodulatory activities at different levels that we believe has important implications for the regulation of anti-tumor immunity as well as response to immunotherapy. Disclosures Vardi: Gilead: Research Funding; Janssen: Honoraria. Ramsay:Roche Glycart AG: Research Funding; MedImmune: Research Funding; Celgene Corporation: Research Funding.

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Recent transcriptomic-based analysis of diffuse large B cell lymphoma (DLBCL) has highlighted the clinical relevance of LN fibroblast and tumor-infiltrating lymphocyte (TIL) signatures within the tumor microenvironment (TME). However, the immunomodulatory role of fibroblasts in lymphoma remains unclear. Here, by studying human and mouse DLBCL-LNs, we identified the presence of an aberrantly remodeled fibroblastic reticular cell (FRC) network expressing elevated fibroblast-activated protein (FAP). RNA-Seq analyses revealed that exposure to DLBCL reprogrammed key immunoregulatory pathways in FRCs, including a switch from homeostatic to inflammatory chemokine expression and elevated antigen-presentation molecules. Functional assays showed that DLBCL-activated FRCs (DLBCL-FRCs) hindered optimal TIL and chimeric antigen receptor (CAR) T cell migration. Moreover, DLBCL-FRCs inhibited CD8 + TIL cytotoxicity in an antigen-specific manner. Notably, the interrogation of patient LNs with imaging mass cytometry identified distinct environments differing in their CD8 + TIL-FRC composition and spatial organization that associated with survival outcomes. We further demonstrated the potential to target inhibitory FRCs to rejuvenate interacting TILs. Cotreating organotypic cultures with FAP-targeted immunostimulatory drugs and a bispecific antibody (glofitamab) augmented antilymphoma TIL cytotoxicity. Our study reveals an immunosuppressive role of FRCs in DLBCL, with implications for immune evasion, disease pathogenesis, and optimizing immunotherapy for patients.