Blood RNA Biomarkers Identify Bacterial and Biofilm Coinfections in COVID-19 Intensive Care Patients.

Purpose: Secondary opportunistic coinfections are a significant contributor to morbidity and mortality in intensive care unit (ICU) patients, but can be difficult to identify. Presently, new blood RNA biomarkers were tested in ICU patients to diagnose viral, bacterial, and biofilm coinfections. Methods: COVID-19 ICU patients had whole blood drawn in RNA preservative and stored at -80°C. Controls and subclinical infections were also studied. Droplet digital polymerase chain reaction (ddPCR) quantified 6 RNA biomarkers of host neutrophil activation to bacterial (DEFA1), biofilm (alkaline phosphatase [ALPL], IL8RB/CXCR2), and viral infections (IFI27, RSAD2). Viral titer in blood was measured by ddPCR for SARS-CoV2 (SCV2). Results: RNA biomarkers were elevated in ICU patients relative to controls. DEFA1 and ALPL RNA were significantly higher in severe versus incidental/moderate cases. SOFA score was correlated with white blood cell count (0.42), platelet count (-0.41), creatinine (0.38), and lactate dehydrogenase (0.31). ALPL RNA (0.59) showed the best correlation with SOFA score. IFI27 (0.52) and RSAD2 (0.38) were positively correlated with SCV2 viral titer. Overall, 57.8% of COVID-19 patients had a positive RNA biomarker for bacterial or biofilm infection. Conclusions: RNA biomarkers of host neutrophil activation indicate the presence of bacterial and biofilm coinfections in most COVID-19 patients. Recognizing coinfections may help to guide the treatment of ICU patients.

Outcomes of heparinized adult veno-arterial extracorporeal membrane oxygenation patients managed with low and high activated partial thromboplastin time targets: A systematic review and meta-analysis.

INTRODUCTION: There are no randomized controlled trials comparing low and high activated partial thromboplastin time (aPTT) targets in heparinized adult veno-arterial (VA) extracorporeal membrane oxygenation (ECMO) patients. Our systematic review and meta-analysis summarized complication rates in adult VA ECMO patients treated with low and high aPTT targets. METHODS: Studies published from January 2000 to May 2022 were identified using Pubmed, Embase, Cochrane Library, and LILACS (Latin American and Caribbean Health Sciences Literature). Studies were included if aPTT was primarily used to guide heparin anticoagulation. For the low aPTT group, we included studies where aPTT goal was ≤60 seconds and for the high aPTT group, we included studies where aPTT goal was ≥60 seconds. Proportional meta-analysis with a random effects model was used to calculate pooled complication rates for patients in the two aPTT groups. RESULTS: Twelve studies met inclusion criteria (5 in the low aPTT group and 7 in the high aPTT group). The pooled bleeding complication incidence for low aPTT studies was 53.6% (95% CI = 37.4%-69.4%, I2 = 60.8%) and for high aPTT studies was 43.8% (95% CI = 21.7%-67.1%, I2 = 91.8%). No studies in the low aPTT group reported overall thrombosis incidence, while three studies in the high aPTT group reported overall thrombosis incidence. The pooled thrombosis incidence for high aPTT studies was 16.1% (95% CI = 9.0%-24.5%, I2 = 13.1%). CONCLUSIONS: Adult ECMO patients managed with low and high aPTT goals appeared to have similar bleeding and other complication rates further highlighting the need for a randomized controlled trial.

In Vitro Analysis of Platelet Adhesion, Aggregation, and Surface GP1bα Expression in Stored Refrigerated Whole Blood: A Pilot Study.

BACKGROUND: Warm, fresh whole blood (WB) has been used by the US military to treat casualties in Iraq and Afghanistan. Based on data in that setting, cold-stored WB has been used to treat hemorrhagic shock and severe bleeding in civilian trauma patients in the United States. In an exploratory study, we performed serial measurements of WB's composition and platelet function during cold storage. Our hypothesis was that in vitro platelet adhesion and aggregation would decrease over time. METHODS: WB samples were analyzed on storage days 5, 12, and 19. Hemoglobin, platelet count, blood gas parameters (pH, Po2, Pco2, and Spo2), and lactate were measured at each timepoint. Platelet adhesion and aggregation under high shear were assessed with a platelet function analyzer. Platelet aggregation under low shear was assessed using a lumi-aggregometer. Platelet activation was assessed by measuring dense granule release in response to high-dose thrombin. Platelet GP1bα levels were measured with flow cytometry, as a surrogate for adhesive capacity. Results at the 3 study timepoints were compared using repeat measures analysis of variance and post hoc Tukey tests. RESULTS: Measurable platelet count decreased from a mean of (163 + 53) × 109 platelets per liter at timepoint 1 to (107 + 32) × 109 at timepoint 3 (P = .02). Mean closure time on the platelet function analyzer (PFA)-100 adenosine diphosphate (ADP)/collagen test increased from 208.7 + 91.5 seconds at timepoint 1 to 390.0 + 148.3 at timepoint 3 (P = .04). Mean peak granule release in response to thrombin decreased significantly from 0.7 + 0.3 nmol at timepoint 1 to 0.4 + 0.3 at timepoint 3 (P = .05). Mean GP1bα surface expression decreased from 232,552.8 + 32,887.0 relative fluorescence units at timepoint 1 to 95,133.3 + 20,759.2 at timepoint 3 (P < .001). CONCLUSIONS: Our study demonstrated significant decreases in measurable platelet count, platelet adhesion, and aggregation under high shear, platelet activation, and surface GP1bα expression between cold-storage days 5 and 19. Further studies are needed to understand the significance of our findings and to what degree in vivo platelet function recovers after WB transfusion.

In Vitro Treatment of Extracorporeal Membrane Oxygenation Coagulopathy with Recombinant von Willebrand Factor or Lyophilized Platelets.

OBJECTIVES: The objective was to compare primary hemostasis between adult ECMO patients and cardiac surgical patients before heparinization and cardiopulmonary bypass. Furthermore, the authors explored whether in vitro treatment of ECMO patient blood samples with recombinant von Willebrand Factor (vWF) or lyophilized platelets improved primary hemostasis in vitro. DESIGN: Prospective cohort study. SETTING: Single academic medical center. PARTICIPANTS: Ten cardiac surgical patients and 8 adult ECMO patients. INTERVENTIONS: Cardiac surgical patients and ECMO patients had blood samples collected, and in vitro platelet thrombus formation was assessed using the ATLAS PST device. The ECMO patients had platelet thrombus formation evaluated at baseline and after in vitro treatment with recombinant vWF or lyophilized platelets, whereas cardiac surgical patients had a single blood sample obtained before heparinization and cardiopulmonary bypass run. MEASUREMENTS AND MAIN RESULTS: Median maximum force (39.7 v 260.2 nN) and thrombus area (0.05 v 0.11) at 5 minutes were lower in untreated ECMO patient samples compared with cardiac surgical patients (p = 0.008 and p < 0.001, respectively). The ECMO patient samples treated with recombinant vWF demonstrated an increase in both platelet maximum force (median value of 222.1 v 39.7 nN) (p = 0.01) and platelet thrombus area (median value of 0.16 v 0.05; p = 0.001). The ECMO patient samples treated with lyophilized platelets demonstrated no increase in platelet maximum force (median value of 193.3 v 39.7 nN; p = 0.18); however, there was a significant increase in platelet thrombus area (median value of 0.13 v 0.05; p = 0.04). CONCLUSIONS: Recombinant vWF and lyophilized platelets may help to restore primary hemostasis in ECMO patients. Future studies should further evaluate the safety and efficacy of these potential therapeutics in ECMO patients.

In vitro comparison of spatiotemporal fibrin clot formation dynamics in plasma treated with different protamine-heparin ratios.

INTRODUCTION: Our study aim was to explore how different protamine-heparin ratios impacted enzymatic coagulation and acellular fibrin clot growth in plasma using an in vitro model. We hypothesized that a low protamine-heparin ratio would be associated with superior fibrin clot growth dynamics. METHODS: We performed an in vitro study using 15 plasma samples from a commercial supplier. Different protamine-heparin ratios were added to each donor plasma sample: low ratio (0.7-1), traditional ratio (1-1), and high ratio (1.3-1) and clot formation dynamics were evaluated using a Thrombodynamics analyzer. Study outcomes were initial clot growth velocity and clot size at 30 min. RESULTS: Plasma samples treated with a one-to-one protamine-heparin ratio had significantly lower mean initial clot growth velocity compared to samples treated with a low protamine-heparin ratio; mean difference -2.3 µm/min (95% CI = -4.0 to -0.7, p = .004). Plasma samples treated with a one-to-one protamine-heparin ratio also had significantly smaller mean clot size at 30 min compared to samples treated with a low protamine-heparin ratio; mean difference -54.0 µm (95% CI = -107.6 to -0.4, p = .048). There were no significant differences in mean initial clot growth velocity or clot size at 30 min between plasma samples treated with a high protamine-heparin ratio and those treated with a one-to-one or low protamine-heparin ratio (all p > .05). CONCLUSIONS: Plasma samples treated with a low protamine-heparin ratio had superior clot growth velocity and larger clot size at 30 min compared to a one-to-one ratio, supporting the notion that a low protamine-heparin ratio may optimize enzymatic coagulation after cardiopulmonary bypass.

Pretransplant angiotensin II type 1-receptor antibodies point to an increase in renal graft sub-intimal fibrosis in living- donor kidney transplant recipients.

The association between anti-AT1Rabs and microvascular injury observed in antibody-mediated rejection has been described in kidney graft Biopsies (KGBx). METHODS: We herein describe the histopathologic findings of KGBx performed during the first year of transplantation (Tx) in 134 patients tested for pre-Tx anti-AT1Rabs in cryopreserved sera (04/2009 to 09/2013). Protocol KGBx before implantation (time-zero), 1 year after Tx and for cause KGBx were included. 21/134 Tx patients were anti-AT1Rab positive (≥17 U/mL); 7/21 experienced acute rejection. For comparison a control group with anti-AT1Rabs <17 U/mL, with (n = 16) and without (n = 31) acute rejection was included. RESULTS: Preimplantation KGBx showed no differences in inflammatory and chronic findings, nor in subintimal fibrosis (25 vs 12.8%, p = .42) between patients with anti-AT1Rabs ≥17 U/mL and those with <17 U/mL. Follow-up KGBx revealed a significantly greater proportion of arterial sub-intimal fibrosis (52.3 vs. 27.6%, p = .049) and extension (15.7 vs. 5.3, p = .015) in anti-AT1Rabs ≥17 U/mL compared to anti-AT1Rabs <17 U/mL KGBx. No differences were observed in microcirculation inflammation, nor in interstitial fibrosis or tubular atrophy between groups. Also, anti-AT1Rabs ≥17 U/mL (ß 10.1, 2.3 to 17.8, p = .012) and more importantly anti-AT1Rabs ≥ 30 U/mL (ß12.1, 3.1 to 20.9, p < .01), were independent risk factors associated with vascular occlusion resulting from sub-intimal fibrosis. CONCLUSION: Our study findings have shown that anti-AT1Rab values ≥17 U/mL are significantly associated to sub-intimal fibrosis and a greater percentage of vessel occlusion in kidney graft biopsies obtained during the first year posttransplant, particularly in coexistence with inflammation and de novo DSA.