Fibrin Strands Will Grow from Soluble Fibrin and Hang Up in an In Vitro Microcirculatory Viscoelastic Model: Is This a Major Cause of COVID-19 Associated Coagulopathy?

Viscoelastic testing (VET) by both TEG and ROTEM has demonstrated hypercoagulability early in corona virus disease 2019 (COVID-19) associated coagulopathy (CAC). Additional VET studies demonstrated fibrinolytic shutdown late in a majority of severely ill COVID-19 patients with an associated elevation of d-dimer. Elevated d-dimer confirms that coagulation, followed by fibrinolysis, has occurred. These findings imply that, during CAC, three enzymes-thrombin, Factor XIIIa and plasmin-must have acted in sequence. However, limitations in standard VET analyses preclude exploration of the earliest phases of clot induction, as well as clot formation and clot dissolution in flowing blood. Herein, we describe a novel method illuminating aspects of this unexplored area. In addition, we created an in vitro blood flow model in which the interactions of thrombin, Factor XIII and plasmin with fibrinogen can be studied, allowing the determination of soluble fibrin (SF), the highly unstable form of fibrin that precedes the appearance of a visible clot. This model allows the determination of the SF level at which fibrin microclots begin to form.

A macrophage attack culminating in microthromboses characterizes COVID 19 pneumonia.

INTRODUCTION: A neutrophilic infiltrate characterizes bacterial pneumonia. Macrophage infiltration is similarly characteristic of the viral pneumonia caused by SARS-CoV-2. These infiltrating macrophages, while phagocytic and capable of engulfing virus laden alveolar cells, are also rich in tissue factor-a thromboplastin. This prothrombotic aspect likely explains how a respiratory virus whose malign effects should be confined to the oropharynx, bronchi and lungs, can cause a panoply of extra-pulmonary organ disorders. Elevated ferritin levels in ICU Covid 19 patients, and elevated acute phase proteins suggest immune overreaction. Elevated d-dimers implicate clotting as well. This evidence links hyperactive innate immunity (macrophage lung infiltrates) with the elevated levels of oligomeric fibrin present in the bloodstream of these patients. METHODS: An in-house assay measuring oligomeric (soluble) fibrin (also referred to as soluble fibrin monomer complexes or SFMC) in whole blood, previously developed for monitoring incipient disseminated intravascular coagulation (DIC) during liver transplantation, was made available to COVID ICU attendings. Since SFMC constitutes the input to intravascular fibrin clots and d-dimer reflects fibrin clot dissolution, it was thought that the two tests, run in tandem along with assays of immune activation, might clarify the frequency and possibly the cause of DIC in patients with severe COVID-19 pneumonia. RESULTS: Classical DIC with intravascular clotting and thrombocytopenia was documented only rarely. However, early in the pandemic shortly after the assay was made available, it identified three patients undergoing acute defibrination. In each patient virtually all of the body's fibrinogen was transformed into SFMC over 3-4 days and deposited somewhere in the vasculature without any gross clots being detected. CONCLUSIONS: Three COVID-19 patients with evidence of a hyperactive immune response (elevated ferritin and acute phase proteins) defibrinated while blood levels of SFMC were being monitored. SFMC levels that were five times higher than normal appeared in the circulation during the defibrination process. SFMC at these levels may precipitate as showers of microclots, damaging heart, kidney, brain, and so forth.

Crotalus atrox venom preconditioning increases plasma fibrinogen and reduces perioperative hemorrhage in a rat model of surgical brain injury.

Perioperative bleeding is a potentially devastating complication in neurosurgical patients, and plasma fibrinogen concentration has been identified as a potential modifiable risk factor for perioperative bleeding. The aim of this study was to evaluate preconditioning with Crotalus atrox venom (Cv-PC) as potential preventive therapy for reducing perioperative hemorrhage in the rodent model of surgical brain injury (SBI). C. atrox venom contains snake venom metalloproteinases that cleave fibrinogen into fibrin split products without inducing clotting. Separately, fibrinogen split products induce fibrinogen production, thereby elevating plasma fibrinogen levels. Thus, the hypothesis was that preconditioning with C. atrox venom will produce fibrinogen spilt products, thereby upregulating fibrinogen levels, ultimately improving perioperative hemostasis during SBI. We observed that Cv-PC SBI animals had significantly reduced intraoperative hemorrhage and postoperative hematoma volumes compared to those of vehicle preconditioned SBI animals. Cv-PC animals were also found to have higher levels of plasma fibrinogen at the time of surgery, with unchanged prothrombin time. Cv-PC studies with fractions of C. atrox venom suggest that snake venom metalloproteinases are largely responsible for the improved hemostasis by Cv-PC. Our findings indicate that Cv-PC increases plasma fibrinogen levels and may provide a promising therapy for reducing perioperative hemorrhage in elective surgeries.

Association of plasma dilution with cardiopulmonary bypass-associated bleeding and morbidity.

OBJECTIVE: To investigate the relationship of cardiopulmonary bypass-associated plasma dilution with blood product transfusion and postoperative morbidity. DESIGN: Retrospective chart review. SETTING: Single academic medical center. PARTICIPANTS: Five hundred forty adults undergoing cardiac surgery between January 4, 2005 and September 19, 2007. INTERVENTIONS: Records were analyzed for demographics, blood volumes (BVs), and fluid balance. Plasma protein concentrations (% of baseline) at the end of bypass were calculated. The lowest and highest quartiles of plasma protein concentration were correlated with blood product administration and postoperative complications. MEASUREMENTS AND MAIN RESULTS: At the end of bypass, calculated plasma protein concentrations ranged from a low of 10% to a high of 111% of baseline. Concentrations below 45% of baseline were associated with increased blood product administration, longer ventilator support, and longer intensive care unit stay. CONCLUSIONS: Patient morbidity and likelihood of transfusion were associated with calculated plasma protein concentrations below 45% of baseline. Bleeding and administered fluids decrease both hematocrit and plasma proteins. Infusion of washed, salvaged blood or red blood cells raises hematocrit, but further dilutes clotting factors. If this dilution is excessive, coagulopathy may ensue. Patients with the smallest BVs are at greatest risk, but dilution can negatively impact patients with large BVs as well if the fluid used for cardiopulmonary bypass prime and anesthesia management represents a significant fraction of total BV.

An algorithm for preventing bypass-associated dilutional (BAD) coagulopathy: Theory and example.

OBJECTIVES: Crystalloid administered during cardiopulmonary bypass may significantly dilute clotting factor concentrations, particularly in low blood volume patients. Should the administered fluid (pump prime plus IVs) drop the clotting factor concentrations below approximately 38%, almost all patients will bleed, heparin levels will be overestimated and excessive neutralizing protamine will be administered. This combination can render blood virtually unclottable. This paper describes algorithms that quantify dilution risk and the maximum fluid that can be safely administered. A confirmatory calculation to prevent excessive protamine administration is also described.

Statistical clues to postoperative blood loss: moving averages applied to medical data.

With the advent of computerized databases, medical data has become easy to accumulate; however, effective use of this data continues to pose significant problems. In other circumstances, smoothing algorithms have been used to uncover non-obvious correlations, trends and relationships in noisy data. We have applied four such algorithms to a large dataset of postoperative blood replacement in cardiopulmonary bypass patients. When applied to this dataset, one of the algorithms proved surprisingly effective. It confirmed several previously observed correlations, and also provided an additional series of counterintuitive and apparently unrelated associations. These associations have been explored in an accompanying paper.

Postoperative bypass bleeding: a bypass-associated dilutional (BAD) coagulopathy?

A number of associations with post-bypass bleeding have been described in the accompanying paper. Herein we hypothesize that dilution is an underlying cause through a malign series of bypass-associated events. Heparinized blood behaves anomalously when diluted. Clotting times first shorten somewhat, then--as the dilution of whole blood approaches 50%--rapidly lengthen to unclottability. During cardiopulmonary bypass, low blood volume patients are at a significant risk of clotting factor dilution which will always be more severe than the level of whole blood dilution. If severe enough, this dilution may lower plasma clotting factors to a critical level and may result in excess protamine administration, secondary to overestimation of heparin. The presence of un-neutralized protamine combined with critically lowered clotting factors leads to marked coagulopathy.

Rapid-SF: a rapid whole-blood screen for soluble fibrin monomer.

Thrombosis accounts for a high proportion of disability and death in the West. Although soluble fibrin (SF) assays have been shown to be good predictors of thrombosis, current quantitative assays are too complex or lengthy to provide timely results, while simpler methods are qualitative and lack sensitivity. We here describe a rapid, new, protamine-based whole-blood screening method for SF which is quantitative and suitable for point-of-care use. Citrated whole blood is mixed with reagent under controlled conditions and the time until development of an SF precipitate is measured. Negative samples remain precipitate-free for 300 seconds. Strongly positive samples develop precipitate in as little as 10 seconds. SF times are mathematically converted to arbitrary SF units (SFU). This Rapid-SF test provides a simple and reliable means of detecting the presence of SF, and is well-suited for whole-blood rapid screening in the emergency department, operating room or clinic.

Control of analyzer slope and intercept in the measurement of packed red cell volume (PCV): part I.

Five multichannel hematology analyzers (four different models) were evaluated to determine the ability of analyzers to correctly measure packed cell volume (PCV) values across the clinically relevant range. A sixth analyzer (fifth model) was subsequently included using archival data. Twenty-two different blood samples with PCVs ranging from 0.2 to 0.6 L/L were run in duplicate on each analyzer. Duplicate reference PCV determinations were also done using the recently described ICSH-recommended reference method. Simultaneously, a simpler "surrogate reference" procedure was performed in duplicate. Analyzer values were compared to ICSH reference and to surrogate reference PCV values using Deming regression analysis. Compared to either reference, all analyzers showed a slope of less than 1.0, thereby overestimating the PCV at 0.2 L/L and underestimating the PCV at 0.6 L/L to some extent. This outcome was surprising. Deming regression slopes would be expected to average at 1.0. Instruments whose slopes are significantly less than 1.0 will show decreased sensitivity to both anemic and polycythemic patients. We know of no inherent reason why multichannel analyzers should behave in this fashion.