Challenges in the diagnosis of thrombotic thrombocytopenic purpura.

INTRODUCTION: Immune-mediated TTP (iTTP) is a rare condition without pathognomonic signs and symptoms. For this reason, the diagnosis of iTTP may be delayed or even missed, with potentially catastrophic consequences. AREAS COVERED: The authors performed an extensive literature review on the diagnosis of iTTP and its challenges combined with their own experience in a referral center for patients with iTTP. EXPERT OPINION: Although a definitive diagnosis of iTTP depends on the ADAMTS13 activity result, timely testing is rarely available at many centers to which patients present. If less complex tests were to become available, they would decrease the chances of late and/or missed diagnoses of iTTP throughout the world. While clinical scores to estimate the likelihood of iTTP exist, they are not well known, and can be misleading if used in the wrong context. Furthermore, the three scoring systems (PLASMIC, Bentley, and French) only moderately correlate with each other, which further complicates the landscape. The existence of these scores and how they should be used in practice is but one opportunity that can be seized through more robust programs to educate nonspecialist clinicians on how to recognize and treat patients with iTTP.

Different-sized extracellular vesicles derived from stored red blood cells package diverse cargoes and cause distinct cellular effects.

BACKGROUND: The formation of extracellular vesicles (EVs) occurs during cold storage of RBCs. Transfusion of EVs may contribute to adverse responses in recipients receiving RBCs. However, EVs are poorly characterized with limited data on whether distinct vesicles are formed, their composition, and potential biological effects. STUDY DESIGN AND METHODS: Stored RBC-derived EVs were purified using protocols that separate larger microvesicle-like EVs (LEVs) from smaller exosome-like vesicles (SEVs). Vesicles were analyzed by electron microscopy, content of hemoglobin, heme, and proteins (by mass spectrometry), and the potential to mediate lipid peroxidation and endothelial cell permeability in vitro. RESULTS: SEVs were characterized by having an electron-dense double membrane whereas LEVs had more uniform electron density across the particles. No differences in hemoglobin nor heme levels per particle were observed, however, due to smaller volumes, SEVs had higher concentrations of oxyHb and heme. Both particles contained antioxidant proteins peroxiredoxin-2 and copper/zinc superoxide dismutase, these were present in higher molecular weight fractions in SEVs suggesting either oxidized proteins are preferentially packaged into smaller vesicles and/or that the environment associated with SEVs is more pro-oxidative. Furthermore, total glutathione (GSH + GSSG) levels were lower in SEVs. Both EVs mediated oxidation of liposomes that were prevented by hemopexin, identifying heme as the pro-oxidant effector. Addition of SEVs, but not LEVs, induced endothelial permeability in a process also prevented by hemopexin. CONCLUSION: These data show that distinct EVs are formed during cold storage of RBCs with smaller particles being more likely to mediate pro-oxidant and inflammatory effects associated with heme.

The females have spoken: A patient-centered national survey on the administration of emergent transfusions with the potential for future fetal harm.

BACKGROUND: Traumatic hemorrhage is the leading cause of preventable death. Early in the resuscitation, only RhD-positive red blood cells are likely to be available, which poses a small risk of causing harm to a future fetus if transfused to an RhD-negative females of childbearing age (CBA), that is, 15 to 49 years old. We sought to characterize how the population, in particular females of CBA, felt about emergency blood administration vis-a-vis potential future fetal harm. METHODS: A national survey was performed using Facebook advertisements in three waves from January 2021 to January 2022. The advertisements directed users to the survey site with seven demographic questions and four questions on accepting transfusion with differing probabilities for future fetal harm (none/any/1:100/1:10,000). Acceptance of transfusion questions were scored on 3-point Likert scale (likely/neutral/unlikely). Only completed responses by females were analyzed. RESULTS: Advertisements were viewed 16,600,430 times by 2,169,805 people with 15,396 advertisement clicks and 2,873 surveys initiated. Most (2,256 of 2,873 [79%]) were fully completed. Majority (2,049 of 2,256 [90%]) of respondents were female. Eighty percent of females (1,645 of 2,049) were of CBA. Most females responded "likely" or "neutral" when asked whether they would accept a lifesaving transfusion if the following risk of fetal harm were present: no risk (99%), any risk (83%), 1:100 risk (85%), and 1:10,000 risk (92%). There were no differences between females of CBA versus non-CBA with respect to the likelihood of accepting lifesaving transfusion with any potential for future fetal harm ( p = 0.24). CONCLUSION: This national survey suggests that most females would accept lifesaving transfusion even with the potential low risk of future fetal harm. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level IV.

Pitfalls in Diagnosing Thrombotic Thrombocytopenic Purpura in Sickle Cell Disease.

Thrombotic thrombocytopenia purpura is characterised by microangiopathic haemolytic anaemia and red cell fragmentation on the peripheral smear, neurological involvement and thrombocytopenia. Diagnosis in the context of sickle cell disease can be challenging due to the inherent haemolytic state and the multitude of other associated complications of the latter. Specifically, fat embolism syndrome characterised by respiratory failure, neurological impairment and thrombocytopenia can be misdiagnosed this way. Confirmation of a diagnosis of thrombotic thrombocytopenic purpura requires demonstration of very low levels (<10%) of the metalloproteinase ADAMTS13 which in fat embolism syndrome is normal. Existing scoring systems used to estimate the pre-test probability for thrombotic thrombocytopenic purpura cannot be applied in patients with sickle cell disease due to the chronic underlying haemolysis. Here, we analyse the diagnostic approach in published cases of thrombotic thrombocytopenic purpura affecting patients with sickle-cell disease. The vast majority of cases were characterised by severe respiratory failure before any other manifestation, a feature of fat embolism syndrome but not of thrombotic thrombocytopenic purpura, and all received red cell transfusion prior to receiving therapeutic plasma exchange. Despite the potential overestimation of the pre-test probability using the existing scoring systems, a large number of cases still scored low. There were no cases with documented low ADAMTS13. In the majority this was not tested, while in the 3 cases that ADAMTS13 was tested, levels were normal. Our review suggests that due to many overlapping clinical and laboratory features thrombotic thrombocytopenic purpura may be erroneously diagnosed in sickle cell disease instead of other complications such as fat embolism syndrome and confirmation with ADAMTS13 testing is essential.

Passenger Lymphocyte Syndrome and Autoimmune Hypothyroidism Following Hematopoietic Stem Cell Transplantation.

We present the case of a 24-year-old male, who received a minor ABO-incompatible allogeneic hematopoietic stem cell transplant (HSCT, blood group O+ ⟶ A+) from an HLA-matched unrelated female donor, as consolidation therapy for relapsed precursor-B-cell acute lymphoblastic leukemia. The donor had a known history of Hashimoto's thyroiditis before HSCT. At day +10 posttransplant, the patient developed severe hemolysis, which required emergent red blood cell exchange. Additionally, about a year posttransplant, he had circulating antithyroglobulin antibodies, decreased free-T4 (fT4) and increased serum thyroid-stimulating hormone (TSH). The potential causes of the posttransplant hemolytic episode and hypothyroidism are discussed. While the hemolysis was worsened by the transfusion of A red blood cells (RBCs) in the context of passenger lymphocyte syndrome, the thyroid dysfunction might be explained by an autoimmune disease transferred from the donor. The case highlights the possibility of several non-relapse-related complications of HSCT occurring in the same patient. It is critical that such adverse outcomes are distinguished from classical graft-versus-host disease (GVHD) for adequate recipient counseling, posttransplant screening, and prompt treatment.

Over-transfusion with blood for suspected hemorrhagic shock is not associated with worse clinical outcomes.

BACKGROUND: We evaluated patient outcomes after early, small volume red blood cell (RBC) transfusion in the setting of presumed hemorrhagic shock. We hypothesized that transfusion with even small amounts of blood would be associated with more complications. STUDY DESIGN AND METHODS: Retrospective review of trauma patients admitted to a Level 1 trauma center between 2016-2021. Patients predicted to require massive transfusion who survived ≥72 h were categorized according to units of RBCs transfused in the first 24 h. A Cox regression model stratified by dichotomized ISS and adjusted for SBP <90 mm Hg and pulse >120 bpm on arrival was used to estimate hazard ratios (HRs) for outcomes of interest. RESULTS: A total of 3121 (24%) received RBC transfusion within the first 24 h. Massive transfusion protocol (MTP) was activated in 38% (1188/3121): 17% received no RBCs, 27.4% 1-3 units, 32.4% 4-9 units, and 22.7% ≥10 units. Mean ISS increased with each category of RBC transfusion. There was no difference in the risk of acute kidney injury (AKI), acute respiratory distress syndrome (ARDS), infection, cardiac arrest, venous thromboembolism or stroke for patients receiving 1-3 units compared to the non-transfused group or 4-9 units group (p > 0.05). Compared to those receiving ≥10 units, the 1-3 units group had a significantly lower risk of AKI, ARDS, and cardiac arrest. DISCUSSION: Early empiric RBC transfusion for presumed hemorrhagic shock may subject patients to potential over-transfusion and end-organ damage. Among patients meeting clinical triggers for MTP, 1-3 units of allogeneic RBCs is not associated with worse outcomes.

From Activated Partial Thromboplastin Time to Antifactor Xa and Back Again.

OBJECTIVES: Monitoring is essential to safe anticoagulation prescribing and requires close collaboration among pathologists, clinicians, and pharmacists. METHODS: We describe our experience in the evolving strategy for laboratory testing of unfractionated heparin (UFH). RESULTS: An intrainstitutional investigation revealed significant discordance between activated partial thromboplastin time (aPTT) and antifactor Xa (anti-Xa) assays, prompting a transition from the former to the latter in 2013. With the increasing use of oral factor Xa inhibitors (eg, apixaban, rivaroxaban, edoxaban, betrixaban), which interfere with the anti-Xa assay, we adapted our protocol again to incorporate aPTT in patients admitted on oral Xa inhibitors who require transition to UFH. CONCLUSIONS: Our experience demonstrates key challenges in anticoagulation and highlights the importance of clinical pathologists in helping health systems adapt to the changing anticoagulation landscape.

Initial experimental experience of triple-knockout pig red blood cells as potential sources for transfusion in alloimmunized patients with sickle cell disease.

BACKGROUND: Blood transfusion remains important in the treatment of patients with sickle cell disease (SCD). However, alloimmunization after blood transfusion is associated with patient morbidity and mortality. Triple-knockout (TKO) pigs (i.e., pigs in which the three known xenoantigens to which humans have anti-pig antibodies have been deleted) may be an alternative source of RBCs for these patients because many humans have no preformed antibodies to TKO pig RBCs (pRBCs). METHODS AND MATERIALS: In an in vitro study, plasma from alloimmunized (n = 12) or non-alloimmunized (n = 12) SCD patients was used to determine IgM/IgG binding to, and CDC of, TKO pRBCs. In an in vivo study, after an estimated 25% of blood volume was withdrawn from two capuchin monkeys, CFSE-labeled TKO pRBCs were transfused. Loss of TKO pRBCs was monitored by flow cytometry, and 7 weeks later, 25% of blood was withdrawn, and CFSE-labeled monkey RBCs were transfused. RESULTS: The in vitro study demonstrated that plasma from neither alloimmunized nor non-alloimmunized SCD patients bound IgM/IgG to, or induced CDC of, TKO pRBCs. In the in vivo study, survival of TKO pRBCs in the two capuchin monkeys was of 5 and 7 days, respectively, whereas after allotransfusion, survival was >28 days. CONCLUSIONS: In conclusion, (1) in the present limited study, no antibodies were detected that cross-reacted with TKO pRBCs, and (2) TKO pigs may possibly be an alternate source of RBCs in an emergency if no human RBCs are available.

Perception of risk in massive transfusion as it relates to fetal outcomes: A survey of surgeons and nurses at one American trauma center.

BACKGROUND: The use of blood products early in the resuscitation of bleeding trauma patients is widely accepted, but made difficult by limited supplies of D- red blood cell (RBC)-containing products. Use of D+ RBC-containing products would alleviate this issue, but could lead to alloimmunization. Risk associated with transfusing D+ RBC in emergency bleeding situations is being reconsidered. The level of concern surrounding emergency transfusion as it relates to future fetal harm was surveyed among surgeons and nurses. METHODS: Faculty and staff in the Departments of Surgery and Nursing were surveyed on the risks of receiving an emergency RBC transfusion and the subsequent potential for fetal harm. Answers were grouped as likely to accept (likely/very likely) or refuse transfusion (unlikely/very unlikely). Participants were compared by sex, and women by child-bearing age, ([15-50 years] vs. [>50 years]). RESULTS: Ninety surveys were initiated with 76 fully completed. Male (n = 39) and female (n = 37) respondents were comparable. Most female respondents (30/37, 81%) were of childbearing age. Overall, both males (38/39, 95%) and females (33/37, 89%; p = .19) were likely to accept a transfusion in an emergency. There was no difference in transfusion acceptance if the risk of fetal harm was presented as 1% (p = .73) or 0.1% (p = .51). Most females (34/37, 92%) were not opposed to transfusion even if there was an unspecified risk of future fetal harm. CONCLUSION: Most of the surgeons and nurses who responded would accept a transfusion in an emergency situation even if it might lead to harming a future fetus.

Civilian walking blood bank emergency preparedness plan.

BACKGROUND: The current global pandemic has created unprecedented challenges in the blood supply network. Given the recent shortages, there must be a civilian plan for massively bleeding patients when there are no blood products on the shelf. Recognizing that the time to death in bleeding patients is less than 2 h, timely resupply from unaffected locations is not possible. One solution is to transfuse emergency untested whole blood (EUWB), similar to the extensive military experience fine-tuned over the last 19 years. While this concept is anathema in current civilian transfusion practice, it seems prudent to have a vetted plan in place. METHODS AND MATERIALS: During the early stages of the 2020 global pandemic, a multidisciplinary and international group of clinicians with broad experience in transfusion medicine communicated routinely. The result is a planning document that provides both background information and a high-level guide on how to emergently deliver EUWB for patients who would otherwise die of hemorrhage. RESULTS AND CONCLUSIONS: Similar plans have been utilized in remote locations, both on the battlefield and in civilian practice. The proposed recommendations are designed to provide high-level guidance for experienced blood bankers, transfusion experts, clinicians, and health authorities. Like with all emergency preparedness, it is always better to have a well-thought-out and trained plan in place, rather than trying to develop a hasty plan in the midst of a disaster. We need to prevent the potential for empty shelves and bleeding patients dying for lack of blood.

Convalescent plasma-mediated resolution of COVID-19 in a patient with humoral immunodeficiency.

Convalescent plasma (CP) is widely used to treat COVID-19, but without formal evidence of efficacy. Here, we report the beneficial effects of CP in a severely ill COVID-19 patient with prolonged pneumonia and advanced chronic lymphocytic leukemia (CLL), who was unable to generate an antiviral antibody response of her own. On day 33 after becoming symptomatic, the patient received CP containing high-titer (ID50 > 5,000) neutralizing antibodies (NAbs), defervesced, and improved clinically within 48 h and was discharged on day 37. Hence, when present in sufficient quantities, NAbs to SARS-CoV-2 have clinical benefit even if administered relatively late in the disease course. However, analysis of additional CP units revealed widely varying NAb titers, with many recipients exhibiting endogenous NAb responses far exceeding those of the administered units. To obtain the full therapeutic benefits of CP immunotherapy, it will thus be important to determine the neutralizing activity in both CP units and transfusion candidates.

Plasma levels of S100A8/A9, histone/DNA complexes, and cell-free DNA predict adverse outcomes of immune thrombotic thrombocytopenic purpura.

BACKGROUND: Immune thrombotic thrombocytopenic purpura (iTTP) is a life-threatening blood disorder, primarily resulting from autoantibodies against ADAMTS13. Infection or inflammation often precedes acute iTTP. However, the association of inflammation and inflammatory mediators with disease severity and outcome of acute iTTP is not fully assessed. OBJECTIVES: Here, we determined plasma levels of S100A8/A9, histone/DNA complexes, citrullinated histone H3 (CitH3), and cell-free DNA (cfDNA) in a cohort of 108 acute episodes from 94 unique iTTP patients and healthy controls, and assessed the association of each of these biomarkers with the disease severity and mortality. RESULTS: All acute iTTP patients had significantly increased plasma levels of S100A8/A9 (median 84.8, interquartile range [IQR] 31.2-157.4 µg/mL), histone/DNA complexes (median 55.7, IQR 35.8-130.8 U/mL), CitH3 (median 3.8, IQR 2.2-6.4 ng/mL), and cfDNA (median 937.7, IQR 781.3-1420.0 ng/mL) on the admission blood samples when compared with healthy controls. An increased plasma level of S100A8/A9, histone/DNA complex and cfDNA was associated with organ damage, coagulopathy, and mortality in iTTP. After being adjusted for age and history of hypertension, Cox proportional hazard regression analysis demonstrated that a hazard ratio (95% confidence interval) for an elevated plasma level of S100A8/A9, histone/DNA complexes, and cfDNA was 11.5 (1.4-90.9) (P = .021), 10.3 (2.7-38.5) (P = .001), and 12.8 (3.9-42.0) (P = .014), respectively. CONCLUSION: These results indicate that inflammation or plasma inflammatory mediators such as S100A8/A9 or NETosis markers such as histone/DNA complexes and cfDNA may play a role in pathogenesis of iTTP, which may help stratify patients with a high risk of death during acute iTTP episodes.

Whole blood transfusion versus component therapy in trauma resuscitation: a systematic review and meta-analysis.

BACKGROUND: Patients with hemorrhagic shock from trauma often require balanced blood product transfusion with red blood cells, plasma, and platelets. Resuscitation with whole blood resuscitation is becoming a common practice. We performed a systematic review and meta-analysis of studies comparing whole blood transfusion with balanced component therapy in patients suffering from traumatic hemorrhagic shock. METHODS: We searched MEDLINE Ovid, EMBASE, and the Cochrane Library for human studies comparing whole blood with component blood therapy published from January 2007 to June 2019. We included studies from both civilian and military settings and that reported 24-hour, in-hospital, or 30-day mortality. We followed the Preferred Reporting Items in Systematic Reviews and Meta-Analyses (PRISMA) guidelines, assessing study quality, publication bias, and heterogeneity. We used meta-analytic models to determine the associations (odds ratio [OR] with 95% confidence interval [CI]) between whole blood transfusion and (1) 24-hour mortality, and (2) in-hospital or 30-day mortality. RESULTS: A total of 1759 identified studies, 12 (reporting on n = 8431 patients) met inclusion criteria. There was heterogeneity in the design, setting, interventions, and outcomes of the studies. On meta-analysis, whole blood transfusion was not associated with 24-hour mortality (OR = 0.83; 95% CI = 0.56-1.24) or in-hospital/30-day mortality (OR = 0.79; 95% CI = 0.48-1.31). CONCLUSION: In this systematic review and meta-analysis, compared with conventional component transfusion, whole blood was not associated with 24-hour or in-hospital mortality. However, there were important limitations with and heterogeneity among the primary studies. Additional study is needed to determine the effectiveness of whole blood.

Damage to red blood cells during whole blood storage.

BACKGROUND: Transfusion with stored whole blood (WB) is increasingly routine practice to resuscitate bleeding trauma patients. Storage of packed red blood cells (pRBC) results in multiple biochemical, structural, and metabolic changes, referred to as to the storage lesion that may mediate adverse effects associated with transfusion of older RBC units. These include increased hemolysis, oxidative stress, and accelerated scavenging of nitric oxide (NO). Whether similar changes occur to stored WB is unclear and are characterized in this study. METHODS: Ten WB units, in citrate-phosphate-dextrose, were purchased from the American Red Cross and changes in hemolysis (increased free hemoglobin, heme, and microparticles), oxidative stress indexed by redox cycling of peroxiredoxin-2 (Prx-2) and NO-scavenging kinetics were determined at different storage times until expiration. RESULTS: Microparticle number and free hemoglobin, but not heme, increased in a storage time-dependent manner. When normalized to the initial number of RBCs in stored WB units, hemolysis rates were similar to those reported for pRBCs. Prx-2 recycling kinetics were slower at expiration compared with earlier storage times. Rates of NO dioxygenation did not change with storage, but were decreased compared with freshly isolated RBCs. CONCLUSION: Storage of WB results in changes associated with the pRBC storage lesion but not for all parameters tested. The relative rate of hemolysis (indexed by free hemoglobin and microparticles) and oxidative stress was similar to that of pRBCs. However, the absolute level of hemolysis products were lower due to lower hematocrit of stored WB units. The clinical significance of these findings requires further investigation.