Off-the-shelf cryopreserved platelets for the detection of HIT and VITT antibodies.

Heparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically simple platelet factor 4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but nonspecific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay end points, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to extend platelet viability significantly by optimizing storage conditions and developed a simple functional assay end point by measuring the release of a platelet α-granule protein, thrombospondin-1 (TSP1), in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several-month-old cryopreserved platelets were treated with PF4 or heparin and were evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated, but not heparin-treated, cryopreserved platelets, consistent with recent data suggesting the requirement for PF4-treated platelets for VITT antibody detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically simple functional testing available for rapid and accurate in-hospital diagnosis.

Management Considerations for Air Medical Transport Programs Transfusing RhD-Positive Red Blood Cell-Containing Products to Females of Childbearing Potential.

Recent years have seen increased discussion surrounding the benefits of damage control resuscitation, prehospital transfusion (PHT) of blood products, and the use of whole blood over component therapy. Concurrent shortages of blood products with the desire to provide PHT during air medical transport have prompted reconsideration of the traditional approach of administering RhD-negative red cell-containing blood products first-line to females of childbearing potential (FCPs). Given that only 7% of the US population has blood type O negative and 38% has O positive, some programs may be limited to offering RhD-positive blood products to FCPs. Adopting the practice of giving RhD-positive blood products first-line to FCPs extends the benefits of PHT to such patients, but this practice does incur the risk of future hemolytic disease of the fetus and newborn (HDFN). Although the risk of future fetal mortality after an RhD-incompatible transfusion is estimated to be low in the setting of acute hemorrhage, the number of FCPs who are affected by this disease will increase as more air medical transport programs adopt this practice. The process of monitoring and managing HDFN can also be time intensive and costly regardless of the rates of fetal mortality. Air medical transport programs planning on performing PHT of RhD-positive red cell-containing products to FCPs should have a basic understanding of the pathophysiology, prevention, and management of hemolytic disease of the newborn before introducing this practice. Programs should additionally ensure there is a reliable process to notify receiving centers of potentially RhD-incompatible PHT because alloimmunization prophylaxis is time sensitive. Facilities receiving patients who have had PHT must be prepared to identify, counsel, and offer alloimmunization prophylaxis to these patients. This review aims to provide air medical transport professionals with an understanding of the pathophysiology and management of HDFN and provide a template for the early management of FCPs who have received an RhD-positive red cell-containing PHT. This review also covers the initial workup and long-term anticipatory guidance that receiving trauma centers must provide to FCPs who have received RhD-positive red cell-containing PHT.

Analysis of College of American Pathologists von Willebrand Factor Proficiency Testing Program.

Von Willebrand factor (VWF) level and/or function is altered in von Willebrand disease (VWD), the most common heritable bleeding disorder worldwide. Laboratory assessment of VWF is continually evolving. Historically, the primary method for the assessment of VWF platelet-binding activity was the ristocetin cofactor assay (VWF:RCo). Contemporary alternative measures of VWF platelet-binding activity include VWF:GPIbR (recombinant; using ristocetin), VWF:GPIbM (recombinant; gain-of-function mutant), and monoclonal antibody. Recently, the American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Federation of Hemophilia collaboration issued guidelines recommending the use of newer assays of VWF platelet-binding activity (VWF: GPIbM, VWF: GPIbR) over VWF:RCo, given known limitations of the VWF:RCo assay. Despite this recommendation, the newer VWF:GPIbM and VWF:GPIbR assays are not United States Food and Drug Administration cleared, limiting their availability in the United States. We sought to assess assay utilization trends, agreement of VWF testing methods, and imprecision of VWF testing (based on assigned sample type) from the College of American Pathologists Proficiency Testing Surveys. The analysis confirms that, while VWF antigen testing has low imprecision, the various VWF activity assays have significant interassay variability, with VWF:RCo showing greater imprecision than the newer GPIb-binding assays. The overall trends in assay utilization reflect the barriers to complete compliance with modern VWD diagnostic guidelines in North America.

Analysis of College of American Pathologists von Willebrand Factor Proficiency Testing Program.

Von Willebrand factor (VWF) level and/or function is altered in von Willebrand disease (VWD), the most common heritable bleeding disorder worldwide. Laboratory assessment of VWF is continually evolving. Historically, the primary method for the assessment of VWF platelet-binding activity was the ristocetin cofactor assay (VWF:RCo). Contemporary alternative measures of VWF platelet-binding activity include VWF:GPIbR (recombinant; using ristocetin), VWF:GPIbM (recombinant; gain-of-function mutant), and monoclonal antibody. Recently, the American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Federation of Hemophilia collaboration issued guidelines recommending the use of newer assays of VWF platelet-binding activity (VWF: GPIbM, VWF: GPIbR) over VWF:RCo, given known limitations of the VWF:RCo assay. Despite this recommendation, the newer VWF:GPIbM and VWF:GPIbR assays are not United States Food and Drug Administration cleared, limiting their availability in the United States. We sought to assess assay utilization trends, agreement of VWF testing methods, and imprecision of VWF testing (based on assigned sample type) from the College of American Pathologists Proficiency Testing Surveys. The analysis confirms that, while VWF antigen testing has low imprecision, the various VWF activity assays have significant interassay variability, with VWF:RCo showing greater imprecision than the newer GPIb-binding assays. The overall trends in assay utilization reflect the barriers to complete compliance with modern VWD diagnostic guidelines in North America.

Hyperhemolysis in a patient with sickle cell disease and recent SARS-CoV-2 infection, with complex auto- and alloantibody work-up, successfully treated with tocilizumab.

BACKGROUND: Hyperhemolysis syndrome (HHS) is a severe delayed hemolytic transfusion reaction seen in sickle cell disease (SCD) patients, characterized by destruction of donor and recipient RBCs. It results in a drop in hemoglobin to below pretransfusion levels and frequently reticulocytopenia. CASE REPORT: We report a case of a man in his thirties with SCD with a recent hospitalization 2 weeks prior for COVID-19. His red cell antibody history included anti-Fy(a) and warm autoantibody. At that time, he was given 2 units of RBC and discharged with a hemoglobin of 10.2 g/dl. He returned to the hospital approximately 1.5 weeks later with hemoglobin 6.0 g/dl and symptoms concerning for acute chest syndrome. Pretransfusion testing now showed 4+ pan-agglutinin in both gel-based and tube-based testing. Alloadsorption identified an anti-N and a strong cold agglutinin. Three least incompatible units were transfused to this patient over several days, with evidence of hemolysis. Further reference lab work revealed anti-Fya , anti-Fyb , anti-Lea , anti-Leb , and an anti-KN system antibody. The patient's hemoglobin nadired at 4.4 g/dl. The patient was treated with a single dose of tocilizumab, his hemoglobin stabilized, and he was discharged. DISCUSSION: We present a case of HHS proximate to recent SARS-CoV-2 infection with multiple allo and autoantibodies identified. Information on the relationship between SARS-CoV-2 infection and HHS is limited; however, it is possible that inflammation related to COVID-19 could predispose to HHS. Tocilizumab is an approved treatment for COVID-19. Additionally, tocilizumab appears to be a promising treatment option for patients with HHS.

Persistence of Ad26.COV2.S-associated vaccine-induced immune thrombotic thrombocytopenia (VITT) and specific detection of VITT antibodies.

Rare cases of COVID-19 vaccinated individuals develop anti-platelet factor 4 (PF4) antibodies that cause thrombocytopenia and thrombotic complications, a syndrome referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). Currently, information on the characteristics and persistence of anti-PF4 antibodies that cause VITT after Ad26.COV2.S vaccination is limited, and available diagnostic assays fail to differentiate Ad26.COV2.S and ChAdOx1 nCoV-19-associated VITT from similar clinical disorders, namely heparin-induced thrombocytopenia (HIT) and spontaneous HIT. Here we demonstrate that while Ad26.COV2.S-associated VITT patients are uniformly strongly positive in PF4-polyanion enzyme-linked immunosorbent assays (ELISAs); they are frequently negative in the serotonin release assay (SRA). The PF4-dependent p-selectin expression assay (PEA) that uses platelets treated with PF4 rather than heparin consistently diagnosed Ad26.COV2.S-associated VITT. Most Ad26.COV2.S-associated VITT antibodies persisted for >5 months in PF4-polyanion ELISAs, while the PEA became negative earlier. Two patients had otherwise unexplained mild persistent thrombocytopenia (140-150 x 103 /µL) 6 months after acute presentation. From an epidemiological perspective, differentiating VITT from spontaneous HIT, another entity that develops in the absence of proximate heparin exposure, and HIT is important, but currently available PF4-polyanion ELISAs and functional assay are non-specific and detect all three conditions. Here, we report that a novel un-complexed PF4 ELISA specifically differentiates VITT, secondary to both Ad26.COV2.S and ChAdOx1 nCoV-19, from both spontaneous HIT, HIT and commonly-encountered HIT-suspected patients who are PF4/polyanion ELISA-positive but negative in functional assays. In summary, Ad26.COV2.S-associated VITT antibodies are persistent, and the un-complexed PF4 ELISA appears to be both sensitive and specific for VITT diagnosis.

Refractory vaccine-induced immune thrombotic thrombocytopenia (VITT) managed with delayed therapeutic plasma exchange (TPE).

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a newly described hematologic disorder, which presents as acute thrombocytopenia and thrombosis after administration of the ChAdOx1 nCov-19 (AstraZeneca) and Ad26.COV2.S (Johnson & Johnson) adenovirus-based vaccines against COVID-19. Due to positive assays for antibodies against platelet factor 4 (PF4), VITT is managed similarly to autoimmune heparin-induced thrombocytopenia (HIT) with intravenous immunoglobulin (IVIG) and non-heparin anticoagulation. We describe a case of VITT in a 50-year-old man with antecedent alcoholic cirrhosis who presented with platelets of 7 × 103 /µL and portal vein thrombosis 21 days following administration of the Ad26.COV2.S COVID-19 vaccine. The patient developed progressive thrombosis and persistent severe thrombocytopenia despite IVIG, rituximab and high-dose steroids and had persistent anti-PF4 antibodies over 30 days after his initial presentation. As such, delayed therapeutic plasma exchange (TPE) was pursued on day 32 of admission as salvage therapy, with a sustained improvement in his platelet count. Our case serves as proof-of-concept of the efficacy of TPE in VITT.

Decreasing blood wastage during ex vivo lung perfusion recovery through utilization of thermal control technology.

BACKGROUND: The Organ Care System (OCS) is a revolutionary ex vivo organ perfusion technology that can potentially expand the organ retrieval range. The OCS Lung device uses packed red blood cells (pRBC) with a proprietary solution. We report the ability to reduce blood waste during this procedure by using a thermal packaging solution in conjunction with the OCS platform. METHODS: We retrospectively reviewed all OCS Lung recoveries performed by our recovery team, using pRBCfrom May 2019 to January 2021. Initially, units were stored using passive refrigeration with the Performance cooler at a temperature range of 1-6°C for 4 h. Subsequently, thermal control technology with the ProMed cooler was utilized to maintain the same temperature range for 72 h. RESULTS: Twenty-three recoveries were initiated with 63 pRBC. The Performance cooler was used for 8, while the ProMed cooler for 13. 37.5% of pRBC transported with the Performance cooler was used within the validated time range, while 25.0% were used beyond the validated time range based on clinical judgment. In addition, 37.5% of pRBC transported with the Performance cooler were returned to the institution after canceled recoveries with an estimated loss of $1800; the ProMed cooler had no wastage. CONCLUSIONS: This study showed that using an advanced thermal packaging solution facilitates proper storage of pRBC and represents an advancement for extended donor lung preservation. The elimination of blood wastage in this initial study portends ongoing benefits for the limited blood supply and reduced cost.

The Impact of COVID-19 Disease on Platelets and Coagulation.

Coronavirus disease 2019 (COVID-19) causes a spectrum of disease; some patients develop a severe proinflammatory state which can be associated with a unique coagulopathy and procoagulant endothelial phenotype. Initially, COVID-19 infection produces a prominent elevation of fibrinogen and D-dimer/fibrin(ogen) degradation products. This is associated with systemic hypercoagulability and frequent venous thromboembolic events. The degree of D-dimer elevation positively correlates with mortality in COVID-19 patients. COVID-19 also leads to arterial thrombotic events (including strokes and ischemic limbs) as well as microvascular thrombotic disorders (as frequently documented at autopsy in the pulmonary vascular beds). COVID-19 patients often have mild thrombocytopenia and appear to have increased platelet consumption, together with a corresponding increase in platelet production. Disseminated intravascular coagulopathy (DIC) and severe bleeding events are uncommon in COVID-19 patients. Here, we review the current state of knowledge of COVID-19 and hemostasis.

Basic principles of viscoelastic testing.

BACKGROUND: Viscoelastic testing is a method of hemostatic analysis that provides a real-time, holistic view of ex vivo clotting. It allows for examination of both cellular and plasma protein contributions to clotting including platelet number and function, fibrin(ogen) function, and coagulation factor function. The method assesses physical clot properties during the transition of blood from a liquid to a gel state, either by measurement of clot shear modulus using physical force transduction or by measurement of clot resonance frequency using sonometric interrogation. Results are reported in a live trace, with different trace parameters reflecting different contributors to hemostasis. These reported parameters vary between testing platforms. RESULTS: In the United States, there are several commonly used Food and Drug Administration (FDA)-approved viscoelastic instruments available on the market. Those instruments that use sonometric clot assessment are more recently available and allow for improved portability for use near the patient's bedside. These instruments generally feature different reagent kits that allow more specific interrogation of different hemostatic pathways. Viscoelastic testing can predict the results of traditional plasma-based coagulation assays and has the added benefit of detecting hypercoagulability and severe hyperfibrinolysis. Implementation of viscoelastic testing in many clinical settings is becoming widespread and has proven to be efficacious in reducing blood transfusion rates in many settings. An impact on overall mortality and morbidity has not yet been demonstrated. CONCLUSION: This article provides a narrative review of the basic principles of viscoelastic testing, including the science and technology behind the method, as well as currently available testing platforms and reagents.

Plasma exchange for heparin-induced thrombocytopenia in patients on extracorporeal circuits: A challenging case and a survey of the field.

Current management of heparin-induced thrombocytopenia (HIT) involves prompt discontinuation of all heparin products and concomitant initiation of a direct thrombin or anti-Xa inhibitor for anticoagulation. In the setting of HIT complicated by an urgent need for cardiopulmonary bypass (CPB), the safety and the efficacy of short-term heparin-based anticoagulation after therapeutic plasma exchange (TPE) have been previously demonstrated. Patients with HIT requiring TPE are frequently on extracorporeal circuits (either CPB, extracorporeal membrane oxygenation [ECMO] or external ventricular assist devices [VADs]). Performing TPE in parallel with these circuits involves additional consideration for circuit size, anticoagulant/citrate management, as well as flow rates, and risk of air embolus. We report a case of a patient with HIT on external biventricular assist device (BiVAD) requiring urgent CPB who experienced thrombotic and hemolytic complications related to anticoagulation management around apheresis line placement for TPE. We also present results from a national survey of academic apheresis services regarding specific practices in managing patients with HIT on extracorporeal circuits who require TPE. In addition, we demonstrate the utility of TPE in patients with HIT on extracorporeal circuits and the risks of this procedure and the need to develop practice guidelines.

Red blood cell exchange in patients with sickle cell disease-indications and management: a review and consensus report by the therapeutic apheresis subsection of the AABB.

BACKGROUND: A prior practice survey revealed variations in the management of patients with sickle cell disease (SCD) and stressed the need for comprehensive guidelines. Here we discuss: 1) common indications for red blood cell exchange (RCE), 2) options for access, 3) how to prepare the red blood cells (RBCs) to be used for RCE, 4) target hemoglobin (Hb) and/or hematocrit (Hct) and HbS level, 5) RBC depletion/RCE, and 6) some complications that may ensue. STUDY DESIGN AND METHODS: Fifteen physicians actively practicing apheresis from 14 institutions representing different areas within the United States discussed how they manage RCE for patients with SCD. RESULTS: Simple transfusion is recommended to treat symptomatic anemia with Hb level of less than 9 g/dL. RCE is indicated to prevent or treat complications arising from the presence of HbS. The most important goals are reduction of HbS while also preventing hyperviscosity. The usual goals are a target HbS level of not more than 30% and Hct level of less than 30%. CONCLUSION: Although a consensus as to protocol details may not be possible, there are areas of agreement in the management of these patients, for example, that it is optimal to avoid hyperviscosity and iron overload, that a target Hb S level in the range of 30% is generally desirable, and that RCE as an acute treatment for pain crisis in the absence of other acute or chronic conditions is ordinarily discouraged.

Acquired factor XI deficiency and therapeutic plasma exchange.

Congenital factor XI (FXI) deficiency is associated with a variable bleeding phenotype. Recent reports have documented the use of therapeutic plasma exchange to rapidly and isovolumetrically increase FXI levels before invasive procedures in patients with congenital FXI deficiency. We report a case of acquired FXI deficiency in a pregnant woman with lupus. We proved that the inhibitor was an IgG, therefore potentially capable of crossing the placenta. While immune suppression eliminated detectable circulating inhibitor, the woman's FXI remained quite low. A multi-disciplinary team was formed and therapeutic plasma exchange with 100% plasma replacement was performed when the patient went into labor, to acutely raise her FXI level and remove any potential non-neutralizing inhibitor. The mother had a controllable level of bleeding during post-TPE cesarean section; the baby had no bleeding and the baby's FXI levels were not overtly abnormal. Therapeutic plasma exchange in acquired FXI deficiency (or other acquired hemophilias) can both acutely isovolumetrically raise factor levels and remove any circulating inhibitor.

The management of anticoagulation in patients undergoing therapeutic plasma exchange: A concise review.

We surveyed multiple apheresis centers represented by the authors for their clinical approach to the management of anticoagulation issues during therapeutic plasma exchange (TPE). We present the results of their practices and a review of the pertinent literature. As plasma is removed during TPE, replacement with all or partial non-plasma-containing fluids (eg, 5% albumin) may lead to significant changes in hemostasis. These changes are amplified in patients who are receiving anticoagulation. We discuss various anticoagulants as well as the monitoring and adjustment of anticoagulation before, during, and after TPE. No single guideline can be applied, but rather, patients must be monitored individually, taking into account their often complex clinical conditions and medication profiles.

Therapeutic plasma exchange as part of multimodal treatment of acquired hemophilia in a patient with concurrent acute intracerebral bleed and pulmonary embolism.

BACKGROUND: Autoantibodies against Factor VIII (FVIII) define the rare but life-threatening bleeding disorder acquired hemophilia A (AHA). Correction of FVIII deficiency and eradication of the factor inhibitor are the ultimate therapeutic goals in this disorder. Bypassing agents such as recombinant factor VIIa (rFVIIa) or FVIII inhibitor bypassing agent are often used to control coagulopathy before the inhibitor is eradicated. Bypassing agents carry a risk of thrombosis, however. CASE REPORT: We report a patient with newly diagnosed AHA and thalamic bleed who additionally had active atrial fibrillation and developed a segmental pulmonary embolism, limiting tolerable rFVIIa dosage. This patient with very-high-risk brain bleed and concurrent thrombosis on bypass agent represented a significant management dilemma for which we successfully utilized therapeutic plasma exchange (TPE) to reduce the inhibitor titer. RESULTS: FVIII inhibitor was undetectable and FVIII level was above the lower limit of normal within 12.5 days from starting TPE. While the patient ultimately died 24 days from admission for reasons unrelated to bleeding, his intracerebral hemorrhage was unchanged in size and no other bleeding morbidity was observed. CONCLUSION: This patient achieved eradication of FVIII inhibitor and did not have bleed expansion while receiving multimodal therapy including corticosteroids, rituximab, and TPE. We discuss the periprocedural risks of TPE in an acquired hemophilia patient and our multiteam management of that risk.