Red blood cells contain enzymatically active GPx4 whose abundance anticorrelates with hemolysis during blood bank storage.

The antioxidant function of the phospholipid hydroperoxide glutathione peroxidase (GPx4) is vital for the homeostasis of many cell types, from neoplastic cells to normal erythroid precursors. However, some functional proteins in erythroid precursors are lost during the development of red blood cells (RBCs); whether GPx4 is maintained as an active enzyme in mature RBCs has remained unclear. Our meta-analyses of existing RBC proteomics and metabolomics studies revealed the abundance of GPx4 to be correlated with lipid-anchored proteins. In addition, GPx4 anti-correlated with lyso-phospholipids and complement system proteins, further supporting the presence of active GPx4 in mature RBCs. To test the potential biological relevance of GPx4 in mature RBCs, we correlated the rate of hemolysis of human RBCs during storage with the abundance of GPx4 and other heritable RBC proteins. Of the molecules that anti-correlated with the rate of hemolysis of RBCs, proteins that mediate the cellular response to hydroperoxides, including GPx4, have the greatest enrichment. Western blotting further confirmed the presence of GPx4 antigenic protein in RBCs. Using an assay optimized to measure the activity of GPx4 in RBCs, we found GPx4 to be an active enzyme in mature RBCs, suggesting that GPx4 protects RBCs from hemolysis during blood bank storage.

Piloting and implementation of quality assessment and quality control procedures in RBC-Omics: a large multi-center study of red blood cell hemolysis during storage.

BACKGROUND: The major aims of the RBC-Omics study were to evaluate the genomic and metabolomic determinants of spontaneous and stress-induced hemolysis during RBC storage. This study was unique in scale and design to allow evaluation of RBC donations from a sufficient number of donors across the spectrum of race, ethnicity, sex, and donation intensity. Study procedures were carefully piloted, optimized, and controlled to enable high-quality data collection. METHODS: The enrollment goal of 14,000 RBC donors across four centers, with characterization of RBC hemolysis across two testing laboratories, required rigorous piloting and optimization and establishment of a quality assurance (QA) and quality control (QC) program. Optimization of WBC elution from leukoreduction (LR) filters, development and validation of small-volume transfer bags, impact of manufacturing and sample-handling procedures on hemolysis parameters, and testing consistency across laboratories and technicians and over time were part of this quality assurance/quality control program. RESULTS: LR filter elution procedures were optimized for obtaining DNA for analysis. Significant differences between standard and pediatric storage bags led to use of an alternative LR-RBC transfer bag. The impact of sample preparation and freezing methods on metabolomics analyses was evaluated. Proficiency testing monitored and documented testing consistency across laboratories and technicians. CONCLUSION: Piloting and optimization, and establishment of a robust quality assurance/quality control program documented process consistency throughout the study and was essential in executing this large-scale multicenter study. This program supports the validity of the RBC-Omics study results and a sample repository that can be used in future studies.

The heritability of hemolysis in stored human red blood cells.

BACKGROUND: The transfusion of red blood cells (RBCs) with maximum therapeutic efficacy is a major goal in transfusion medicine. One of the criteria used in determining stored RBC quality is end-of-storage hemolysis. Between donors, a wide range of hemolysis is observed under identical storage conditions. Here, a potential mechanism for this wide range is investigated. We hypothesize that the magnitude of hemolysis is a heritable trait. Also, we investigated correlations between hemolysis and RBC metabolites; this will establish pathways influencing hemolysis as future targets for genetic analysis. STUDY DESIGN AND METHODS: Units of RBCs from identical and nonidentical twins were collected and stored under standard conditions for 56 days. Hemolysis, adenosine triphosphate (ATP), and total glutathione (tGSH) were measured throughout storage. Nontargeted metabolic analyses were performed on RBCs that had been stored for 28 days. Heritability was determined by comparing values between identical and nonidentical twins. RESULTS: Hemolysis was found to be heritable (mean > 45%) throughout the storage period. Potential correlations were observed between hemolysis and metabolites from the purine metabolism, lysolipid, and glycolysis pathways. These also exhibited heritability (>20%). No correlation was found with ATP or tGSH. CONCLUSION: The susceptibility of RBCs to lysis during storage is partly determined by inheritance. We have also uncovered several pathways that are candidate targets for future genomewide association studies. These findings will aid in the design of better storage solutions and the development of donor screening tools that minimize hemolysis during storage.

Heritability of glutathione and related metabolites in stored red blood cells.

Red blood cells (RBCs) collected for transfusion deteriorate during storage. This deterioration is termed the "RBC storage lesion." There is increasing concern over the safety, therapeutic efficacy, and toxicity of transfusing longer-stored units of blood. The severity of the RBC storage lesion is dependent on storage time and varies markedly between individuals. Oxidative damage is considered a significant factor in the development of the RBC storage lesion. In this study, the variability during storage and heritability of antioxidants and metabolites central to RBC integrity and function were investigated. In a classic twin study, we determined the heritability of glutathione (GSH), glutathione disulfide (GSSG), the status of the GSSG,2H(+)/2GSH couple (Ehc), and total glutathione (tGSH) in donated RBCs over 56 days of storage. Intracellular GSH and GSSG concentrations both decrease during storage (median net loss of 0.52 ± 0.63 mM (median ± SD) and 0.032 ± 0.107 mM, respectively, over 42 days). Taking into account the decline in pH, Ehc became more positive (oxidized) during storage (median net increase of 35 ± 16 mV). In our study population heritability estimates for GSH, GSSG, tGSH, and Ehc measured over 56 days of storage are 79, 60, 67, and, 75%, respectively. We conclude that susceptibility of stored RBCs to oxidative injury due to variations in the GSH redox buffer is highly variable among individual donors and strongly heritable. Identifying the genes that regulate the storage-related changes in this redox buffer could lead to the development of new methods to minimize the RBC storage lesion.

The heritability of metabolite concentrations in stored human red blood cells.

BACKGROUND: The degeneration of red blood cells (RBCs) during storage is a major issue in transfusion medicine. Family studies in the 1960s established the heritability of the RBC storage lesion based on poststorage adenosine triphosphate (ATP) concentrations. However, this critical discovery has not been further explored. In a classic twin study we confirmed the heritability of poststorage ATP concentrations and established the heritability of many other RBC metabolites. STUDY DESIGN AND METHODS: ATP concentrations and metabolomic profiles were analyzed in RBC samples from 18 twin pairs. On samples stored for 28 days, the heritability of poststorage ATP concentrations were 64 and 53% in CP2D- and AS-3-stored RBCs, respectively. RESULTS: Metabolomic analyses identified 87 metabolites with an estimated heritability of 20% or greater. Thirty-six metabolites were significantly correlated with ATP concentrations (p ≤ 0.05) and 16 correlated with borderline significance (0.05 ≤ p ≤ 0.10). Of the 52 metabolites that correlated significantly with ATP, 24 demonstrated 20% or more heritability. Pathways represented by heritable metabolites included glycolysis, membrane remodeling, redox homeostasis, and synthetic and degradation pathways. CONCLUSION: We conclude that many RBC metabolite concentrations are genetically influenced during storage. Future studies of key metabolic pathways and genetic modifiers of RBC storage could lead to major advances in RBC storage and transfusion therapy.

The concentration of glutathione in human erythrocytes is a heritable trait.

Glutathione (GSH) is a ubiquitous, redox-active, small molecule that is critical to cellular and organism health. In red blood cells (RBCs), the influence of the environment (e.g., diet and lifestyle) on GSH levels has been demonstrated in numerous studies. However, it remains unknown if levels of GSH are determined principally by environmental factors or if there is a genetic component, i.e., heritability. To investigate this we conducted a twin study. Twin studies are performed by comparing the similarity in phenotypes between mono- and dizygotic twin pairs. We determined the heritability of GSH, as well as its oxidation product glutathione disulfide (GSSG), the sum of GSH equivalents (tGSH), and the status of the GSSG/2GSH couple (marker of oxidation status, Ehc) in RBCs. In our study population we found that the estimated heritability for the intracellular concentration of GSH in RBCs was 57 %; for GSSG it was 51 %, tGSH 63 %, and Ehc 70 %. We conclude that a major portion of the phenotype of these traits is controlled genetically. We anticipate that these heritabilities will also be reflected in other cell types. The discovery that genetics plays a major role in the innate levels of redox-active species in RBCs is paradigm shifting and opens new avenues of research in the field of redox biology. Inherited RBC antioxidant levels may be important disease modifiers. By identifying the relative contributions of genes and the environment to antioxidant variation between individuals, new therapeutic strategies can be developed. Understanding the genetic determinants of these inherited traits may allow personalized approaches to relevant therapies.

Severe autoimmune neutropenia associated with acute autoimmune hepatitis.

A 49-year-old previously healthy female presented with acute hepatitis and severe neutropenia. A diagnosis of type 1 autoimmune hepatitis was made based on the histological appearance of a liver core biopsy, positive anti-smooth muscle antibodies, and positive anti-neutrophil cytoplasmic antibody (atypical ANCA). Hemogram revealed mild leukopenia with severe neutropenia (absolute neutrophil count 256/mm(3)), normal hemoglobin and mild thrombocytopenia (115000/mm(3)). A bone marrow biopsy and aspirate had a normal karyotype, increase in granulopoiesis, prominence of promyelocytes (31%) and absence of mature granulocytes. Anti-neutrophil antibodies were detected in the patient's blood. Therapy was directed at the underlying hepatitis with resolution of neutropenia without the use of colony-stimulating factors.

Transfusion-related acute lung injury (TRALI): current clinical and pathophysiologic considerations.

Transfusion-related acute lung injury (TRALI) is a rare transfusion reaction presenting as respiratory distress during or after transfusion of blood products. TRALI varies in severity, and mortality is not uncommon. TRALI reactions have equal gender distributions and can occur in all age groups. All blood products, except albumin, have been implicated in TRALI reactions. TRALI presents as acute respiratory compromise occurring in temporal proximity to a transfusion of a blood product. Other causes of acute lung injury should be excluded in order to definitively diagnose TRALI. Clinically and pathologically, TRALI mimics acute respiratory distress syndrome (ARDS), with neutrophil-derived inflammatory chemokines and cytokines believed to be involved in the pathogenesis of both entities. Anti-HLA and anti-neutrophil antibodies have been implicated in some cases of TRALI. Treatment for TRALI is supportive; prevention is important. It is suspected that TRALI is both underdiagnosed and underreported. One of the difficulties in the evaluation of potential TRALI reactions is, until recently, the lack of diagnostic criteria. A group of transfusion medicine experts, the American-European Consensus Conference (AECC), recently met and developed diagnostic criteria of TRALI, as well as recommendations for management of donors to prevent future TRALI reactions. In light of the AECC consensus recommendations, we report an incident of TRALI in an oncology patient as an example of the potential severity of the lung disease and the clinical and laboratory evaluation of the patient. We also review the literature on this important complication of blood transfusion that internists may encounter.

Platelets photochemically treated with amotosalen HCl and ultraviolet A light correct prolonged bleeding times in patients with thrombocytopenia.

BACKGROUND: Photochemical treatment (PCT) with amotosalen HCl with ultraviolet A illumination inactivates pathogens and white blood cells in platelet (PLT) concentrates. STUDY DESIGN AND METHODS: In a Phase II crossover study, 32 patients with thrombocytopenia received one transfusion of PCT and/or one transfusion of untreated (reference) apheresis PLTs. Hemostatic efficacy was assessed with the cutaneous template bleeding time and clinical observations. RESULTS: Paired bleeding time data for PCT and reference transfusions were available for 10 patients. Mean pretransfusion bleeding times were 29.2 +/- 1.6 minutes in the PCT group and 28.7 +/- 2.5 minutes in the reference group. After transfusion of a dose of PLTs of at least 6.0 x 10(11), mean 1-hour posttransfusion template bleeding times corrected to 19.3 +/- 9.5 minutes in the PCT group and 14.3 +/- 6.5 minutes in the reference group (p = 0.25). In 29 patients receiving paired PCT and reference transfusions, mean 1-hour posttransfusion PLT count increments were 41.9 x 10(9) +/- 20.8 x 10(9) and 52.3 x 10(9) +/- 18.3 x 10(9) per L for PCT and reference, respectively (p = 0.007), and mean 1-hour posttransfusion PLT corrected count increments (CCIs) were 10.4 x 10(3) +/- 4.9 x 10(3) and 13.6 x 10(3) +/- 4.3 x 10(3) for PCT and reference, respectively (p < 0.001). The time to next PLT transfusion was 2.9 +/- 1.2 days after PCT transfusions versus 3.4 +/- 1.3 days after reference transfusions (p = 0.18). Clinical hemostasis was not significantly different after PCT and reference transfusions. CONCLUSION: PCT PLTs provided correction of prolonged bleeding times and transfusion intervals not significantly different than reference PLTs despite significantly lower PLT count increments and CCIs.

Thienopyridine-linked thrombotic microangiopathy: association with endothelial cell apoptosis and activation of MAP kinase signalling cascades.

The thienopyridine platelet antagonist ticlopidine is associated with development of thrombotic thrombocytopenic purpura (TTP) but the pathophysiology of this link is unclear. Severe deficiency of disintegrin and metalloproteinase with thrombospondin motif-13 (ADAMTS13), described in familial cases and a significant fraction of idiopathic TTP, has been reported in only a few ticlopidine-linked cases. As ticlopidine can disrupt production of extracellular matrix (ECM) components critical to microvascular endothelial cell (MVEC) integrity in vitro, we explored the hypotheses that ticlopidine and ticlopidine-linked TTP plasmas induce MVEC apoptosis in a manner similar to that of idiopathic TTP plasmas, and that ECM components and related mitogen-activated protein kinase (MAPK) signalling cascades may be involved in this process. Replicating the activity of plasmas from patients with idiopathic TTP, plasma from five ticlopidine-linked TTP patients induced apoptosis of primary human dermal, glomerular and hepatic MVEC, but had no effect on pulmonary MVEC or large vessel endothelial cells (EC). Pharmacological levels of ticlopidine initiated apoptosis with similar EC lineage restriction. In parallel, ticlopidine and plasmas from idiopathic and ticlopidine-TTP patients decreased transcripts for the ECM component thrombospondin-1 in MVEC, but not in large vessel EC. These changes were accompanied by prolonged induction of MAPKs extracellular signal-related kinase (ERK)-1/2 and p38 only in TTP susceptible MVEC. Induction of apoptosis by ticlopidine and TTP plasma was abrogated by inhibitors of ERK-1/2 and p38 phosphorylation. In conclusion, MVEC apoptosis related to altered ECM-MVEC interactions may be a key part of the pathology of ticlopidine-linked and idiopathic TTP.

Chronic thrombotic microangiopathy associated with antineoplastic therapy with minimal hematologic effects.

OBJECTIVE: To describe 6 patients who developed progressive renal failure and renal thrombotic microangiopathy (TM) not accompanied by the characteristic hematologic disturbances of TM syndromes. PATIENTS AND METHODS: Portions of renal biopsy specimens from each patient were examined by light and electron microscopy for histopathologic evidence of TM. Antecedent clinical events, laboratory evidence of hemolysis and thrombocytopenia, and clinical outcome were documented. Medical records were reviewed and clinical data, including laboratory values, treatment, and outcome, were recorded. RESULTS: In each case, a slowly progressive uremia evolved after radiation and/or chemotherapy without laboratory evidence of acute hemolysis or thrombocytopenia. Renal biopsy specimens in all cases showed TM and tubulointerstitial scarring, suggesting both acute and chronic renal injury. Two of the 6 patients underwent plasma exchange therapy without improvement of renal function. Three patients treated with angiotensin-converting enzyme inhibitors for coexisting systemic hypertension remained stable or had mild improvement in renal function. CONCLUSIONS: A small subset of patients treated for malignancy developed slowly evolving uremia associated with renal TM without marked hematologic abnormalities. In the absence of thrombocytopenia and other typical laboratory findings, the diagnosis of renal TM may be overlooked.

Retinoic acid exacerbates experimental radiation nephropathy.

Studies have shown that angiotensin-converting enzyme inhibitors and an angiotensin II receptor blocker can delay, but cannot reverse, the progression of experimentally induced radiation nephropathy. In an effort to find a method for reversing injury, three agents were tested in a rat model of radiation nephropathy. Pirfenidone (a phenyl-pyridone antifibrotic) and thiaproline (an inhibitor of collagen deposition) were not capable of retarding the development of radiation nephropathy. However, all-trans retinoic acid (an anti-inflammatory agent) exacerbated radiation nephropathy. We speculated that the detrimental effects of retinoic acid might be the result of stimulation of renal cell proliferation. However, retinoic acid had no effect on tubular or glomerular cell proliferation in normal animals and did not enhance radiation-induced proliferation. A recent report that retinoic acids inhibit nitric oxide production suggested an alternative mechanism, since inhibition of production of nitric oxide is known to exacerbate radiation nephropathy. Experiments demonstrated that retinoic acid exacerbated the radiation-induced drop in renal production of nitric oxide, suggesting that the detrimental effect of all-trans retinoic acid might be explained by inhibition of renal nitric oxide activity. Particularly in view of the recent clinical report of enhancement of radiation nephropathy by retinoic acid in patients receiving bone marrow transplantation, the combination of retinoic acid and renal irradiation should be carried out with great caution.