Molecular communication between red blood cells of different phenotypes during blood storage in mice.

BACKGROUND: The cellular and molecular changes during red blood cell (RBC) storage that affect posttransfusion recovery (PTR) remain incompletely understood. We have previously reported that RBCs of different storage biology cross-regulate each other when stored together (co-storage cross-regulation [CSCR]). However, the mechanism of CSCR is unclear. In the current study, we tested the hypothesis that CSCR involves acquisition of molecular signatures associated with PTR. STUDY DESIGN AND METHODS: The whole blood compartment of either B6 or FVB mice was biotinylated in vivo prior to blood collection and storage. Bio-B6 or Bio.FVB were stored with RBCs from B6 mice transgenic for green florescent protein (GFP) (B6.GFP). After storage, avidin-magnetic beads were used to simultaneous purify Bio-RBCs (positive selection) and B6.GFPs (negative selection). Isolated populations were analyzed by transfusion to establish PTR, and subjected to metabolomic and proteomic analysis. RESULTS: B6 RBCs acquired molecular signatures associated with stored FVB RBCs at both the metabolomic and proteomic level including metabolites associated with energy metabolism, oxidative stress regulation, and oxidative damage. Mitochondrial signatures were also acquired by B6 RBCs. Protein signatures acquired by B6 RBCs include proteins associated with vesiculation. CONCLUSION: The data presented herein demonstrate the appearance of multiple molecular changes from poor-storing RBCs in good-storing RBCs during co-storage. Whether this is a result of damage causing intrinsic molecular changes in B6 RBCs or if molecules of FVB RBC origin are transferred to B6 RBCs remains unclear. These studies broaden our mechanistic understanding of RBC storage (in particular) and potentially RBC biology (in general).

In vivo genotoxicity assessment of N-(-9 acridinyl)-b-alanine hydrochloride (S-300) using a validated Pig-a mutagenesis assay.

BACKGROUND: N-(-9 acridinyl)-b-alanine hydrochloride (S-300) is the main byproduct of red blood cell (RBC) amustaline/glutathione(GSH) pathogen reduction, currently undergoing phase III US clinical trials following successful European studies(1-3). Phosphatidylinositol glycan, class A (Pig-a) X-linked gene mutagenesis is a validated mammalian in vivo mutation assay for genotoxicity, assessed as clonal loss of glycosylphosphatidylinositol-linked CD59 cell-surface molecules on reticulocytes (RETs) and RBCs. METHODS: Male Sprague-Dawley rats received continuous infusion of S-300 up to the maximum feasible dose (240 mg/kg/day-limited by solubility and volume) for 28 days. Positive controls received a known mutagen by oral gavage on Days 1-3. Plasma levels of S-300 were assessed by HPLC before, during and after infusion. CD59-negative RBCs and RETs were enumerated in pre-dose and Day 28 samples, using a flow cytometric method. Outcome was evaluated by predetermined criteria using concurrent and historical controls. Toxicity was assessed by laboratory measures and necropsy. RESULTS: S-300 reached maximum, dose-dependent levels (3-15 µmol/L) within 2-8 h that were sustained for 672 h and undetectable 2 h after infusion. Circulating RET levels indicated a lack of hematopoietic toxicity. Necropsy revealed minimal-mild observations related to poor S-300 solubility at high concentrations. Pig-a assessment met the preset acceptability criteria and revealed no increase in mutant RBCs or RETs. CONCLUSIONS: Maximum feasible S-300 exposure of rats by continuous infusion for 28 days was not genotoxic as assessed by an Organization for Economic Cooperation and Development-compliant, mammalian, in vivo Pig-a gene mutation assay that meets the requirements of International Conference on Harmonization (ICH) S2(R1) and FDA guidances on genotoxicity testing.

Red cell transfusion thresholds in outpatients with myelodysplastic syndromes: Results of a pilot randomized trial RBC-ENHANCE.

BACKGROUND: The optimal hemoglobin (Hb) threshold for red blood cell transfusions in adult patients with myelodysplastic syndromes (MDS) has not been defined. STUDY DESIGN AND METHODS: We conducted a pilot randomized multi-center study of two transfusion algorithms (liberal, to maintain Hb 110-120 g/L, transfuse 2 units if Hb < 105 g/L and 1 unit if Hb 105-110 g/L vs. restrictive, 85-105 g/L, transfuse 2 units when Hgb < 85 g/L). Primary objectives were 70% compliance in maintaining the q2 week hemoglobin within the targeted range and the achievement of a 15 g/L difference in pre-transfusion Hb. Secondary outcomes included measures of quality of life (QOL), iron studies and safety. RESULTS: Twenty-eight patients were randomized between February 2015-2020, 13 to the restrictive arm and 15 to the liberal arm in three tertiary care centers. The compliance was 66% and 45% and the mean pre-transfusion Hb thresholds were 86 (standard deviation [SD] 8) and 98 g/L (SD 10) in the restrictive and liberal arms, (mean difference 11.8 g/L, p < .0001), respectively. Patients in the liberal arm experienced a mean of 3.4 (SD 2.6) more transfusion visits and received a mean of 5.3 (SD 5.5) more units of blood during the 12-week study. Ferritin increased by 1043 (SD 1516) IU/L and 148 (SD 1319) IU/L in the liberal and restrictive arms, respectively. Selected QOL scores were superior pre-transfusion and more patients achieved clinically important improvements in the liberal arm compared with the restrictive arm for selected symptoms and function domains. CONCLUSION: The results establish that policies for transfusion support can be delivered in practice at multiple hospitals, but further research is required to understand the full clinical effects and safety of liberal transfusion policies in MDS outpatients.

Reducing unnecessary premedication prior to parenteral iron therapy: A quality improvement project.

BACKGROUND: Intravenous (IV) iron carries risks of mild, self-limiting, tryptase-negative Fishbane and complement activation-related pseudo-allergy reactions, with rare reports of anaphylaxis. Historically, high-molecular-weight iron dextran (HMWID) was associated with a higher incidence of anaphylaxis and empiric premedication with antihistamines/corticosteroids have been used to mitigate this risk. HMWID is no longer available and the risk of hypersensitivity reactions with newer IV iron formulations is low. Therefore, the use of routine prophylactic premedication in all patients is not justified but should be considered in high-risk patients. STUDY DESIGN AND METHODS: Our primary aim was to reduce inappropriate premedication before IV iron administration by 50% so that our institution's hematology providers only prescribe premedications to patients at high risk of having a severe reaction. Interventions included a multidisciplinary education initiative to highlight current evidence against universal administration of premedications and revision of the IV iron informed consent form and electronic order set. RESULTS: We measured the success of our intervention by comparing data collected during a 6-month pre-intervention period (837 infusions) to a 6-month post-intervention period (947 infusions). Inappropriate administration of premedications decreased from 79% in the pre-intervention period compared to 65% in the post-intervention period. We found no significant difference in the number of Fishbane reactions, severe reactions, and emergency room admissions, despite this reduction in premedication use. DISCUSSION: Although we did not reach our goal of a 50% reduction in inappropriate premedication use, opportunities for process improvements were uncovered and are being explored in the next cycle of this quality improvement project.

Successful therapeutic plasma exchange for a patient with sickle cell disease and fat embolism syndrome after a failure of a response to red cell exchange transfusion.

BACKGROUND: Fat embolism syndrome (FES) is a rare complication, which was reported mostly with milder forms of heterozygous sickle cell disease (SCD). It may present in a catastrophic way with multi-organ failure, particularly involving the pulmonary and neurological systems. Diagnosis is often missed or delayed; and the standard recommended treatment is red cell exchange (RCE) transfusion, which has sub-optimal results, such as debilitating long-term neurological complications. Recently, few reports suggested that the addition of Therapeutic Plasma Exchange (TPE) might further improve the outcome. CASE DESCRIPTION: A 23-year-old woman with homozygote SCD was admitted with bony pains and vaso-occlusive crises. However, her course evolved to respiratory failure requiring mechanical ventilation, decreased level of consciousness, skin rash, severe anemia and thrombocytopenia and a picture consistent with thrombotic microangiopathy. MRI of the brain showed scattered multi-focal ischemic foci and cytotoxic edema. The patient received RCE on the third day after admission without improvement. On the seventh day, TPE was instituted (2 L/day of fresh frozen plasma for 5 days), following which she regained her consciousness and showed an improvement in her laboratory abnormalities. On follow up, she had gradual full neurological recovery and resolution of the MRI findings within a few months. CONCLUSION: FES remains a diagnostic and therapeutic challenge, with significant morbidity and mortality. Success in the management of this reported case with the addition of TPE to RCE supports the notion that TPE may be a potentially helpful modality that deserves further research.

The experience of flow cytometry for specific antibody against cisplatin-treated red blood cells: A case report.

BACKGROUND: Cisplatin-associated hemolysis is a rare but important adverse effect. Nonimmunological protein adsorption (NIPA) due to erythrocyte membrane modification has been reported as the leading cause of cisplatin-associated hemolysis. However, limited data exist on cisplatin-associated immunological hemolysis because of a lack of an established diagnostic method. Here, we used flow cytometry (FCM) to diagnose a patient with cisplatin-associated immunological hemolysis. STUDY DESIGN AND METHODS: A 55-year-old woman with uterocervical cancer was treated with weekly cisplatin monotherapy (40 mg/m2 ). She had no previous transfusion and medication history, nor any significant family history. On the 26th day after cisplatin administration, severe hemolysis was noted. Her red blood cells (RBCs) and sera were evaluated by direct antiglobulin test (DAT) and indirect antiglobulin test (IAT), respectively. To explore immunological reactions for cisplatin-treated RBCs, we attempted FCM using cisplatin-treated and -untreated RBCs. After incubating conditioned RBCs with the patient's serum or healthy donor serum, we evaluated their fluorescent intensity by fluorescein isothiocyanate (FITC)-conjugated anti-human immunoglobulin (Ig) G antibodies. RESULTS: The patient's DAT was positive, and an IAT using her plasma was positive for cisplatin-treated RBCs. FCM using cisplatin-treated RBCs revealed that the patient's serum had higher FITC intensity than the donor's serum, indicating the existence of cisplatin-treated RBC-specific IgGs in patient's serum. CONCLUSION: Here, we report a rare case of a patient with hemolysis diagnosed using FCM to identify specific antibodies against cisplatin-treated RBCs. NIPA and immunological mechanisms may contribute to hemolysis onset during cisplatin treatment.

A single center experience for clinical evaluation of paroxysmal cold hemoglobinuria and Donath-Landsteiner testing.

BACKGROUND: Paroxysmal cold hemoglobinuria (PCH) is a rare form of autoimmune hemolytic anemia (AIHA), mainly affecting children. The diagnosis and management are challenging due to similarities to other causes for AIHA and limited availability to Donath-Landsteiner (DL) testing. STUDY DESIGN AND METHODS: In this single-center retrospective study, we aimed to characterize the clinical presentation and outcomes of PCH patients, defined as having positive Donath-Landsteiner antibodies, compared to a cohort of AIHA patients. RESULTS: DL-positive patients were observed to have higher lactate dehydrogenase levels and lower reticulocyte counts compared to DL-negative patients, although this was not statistically significant. We also observed that using steroids in DL-positive patients did not significantly impact their recovery. DISCUSSION: Our findings support the limited published data on PCH patients and further prompt larger multicenter studies to further characterize these patients so that they are more readily identified, especially in centers where DL antibody testing is not readily available.

Racial differences in red blood cell transfusion in hospitalized patients with anemia.

BACKGROUND: Guidelines recommend transfusion of red blood cells (RBC's) when a hospitalized patient's hemoglobin (Hb) drops below a restrictive transfusion threshold, either at 7 or 8 g. Hospitals have implemented transfusion policies to encourage compliance with guidelines and reduce variation in transfusion practice. However, variation in transfusion practice remains. The purpose of this study was to examine whether there is variation in the receipt of transfusion by patient race. METHODS: Hospitalized general medicine patients with anemia (Hb < 10 g/dL) were eligible. Chi-squared tests were used to compare the percent of patients receiving a transfusion by race overall and within strata of their nadir Hb. Linear regression was used to test the association between a patient's race, their nadir Hb, receipt of an RBC transfusion, and the number of units transfused. RESULTS: Four thousand nine hundred and fifty-one patients consented, including 1363 (28%) who received a transfusion. 71% of patients were African American, 25% were White, and 4% were Other Race. Overall African Americans were less likely to be transfused compared to Whites (25% vs. 30%, p < .01), and within Hb strata below a Nadir Hb of 9 g/dL (Hb 8.0-8.9 g/dL 1% vs. 7%, p < .01; 7.0-7.9 g/dL 15% vs. 28%, p < .01; <7 g/dL 80% vs. 86%, p < .01). African Americans also received fewer units of RBC's (ß = -.17, p < .01) overall and at lower Hb levels (ß = .14, p < .01) compared to Whites. DISCUSSION: The Hb level at which patients are transfused at and the total number of RBC units received during hospitalization differ by patient race.

Process and procedural adjustments to improve CD34+ collection efficiency of hematopoietic progenitor cell collections in sickle cell disease.

BACKGROUND: Adequate CD34+ collection efficiency (CE) is critical to achieve target CD34+ cell doses in hematopoietic progenitor cell (HPC) collections. Autologous HPC collection in sickle cell disease (SCD) is associated with unstable collection interfaces and low CD34+ CEs. We hypothesized that variables specific to SCD, activation of blood cells and elevated viscosity, might contribute to these issues and made adjustments to the collection process and procedure to address our hypothesis. STUDY DESIGN AND METHODS: In two patients with SCD undergoing autologous HPC collection on our clinical trial (NCT02193191), we therefore implemented adjustments to the process and procedure in the following areas: proximity of RBC exchange to HPC collection, the type of anticoagulation, and the packing factor setting. RESULTS: There was no collection interface instability. Our CD34+ CE1s were high at 70% and 51%, and granulocyte CE, platelet CE, and product granulocyte % were remarkably low. Product hematocrits were not as high as previously reported to be required to obtain adequate CEs. Interestingly, one HPC product showed a hemoglobin S (HbS) of 91% at the same time that the peripheral blood (PB) showed a HbS of 22%. DISCUSSION: Adjustments to the HPC collection process and procedure were associated with adequate CD34+ CEs and low granulocyte and platelet contamination in HPC products from SCD patients. Given the discrepancy in the percentage of sickle RBCs in the product versus the PB, we hypothesize that CD34+ cells and RBCs may aggregate. Our interventions and hypothesis should be further investigated in larger studies.

Effects of hypoxic storage on the efficacy of gamma irradiation in abrogating lymphocyte proliferation and on the quality of gamma-irradiated red blood cells in additive solution 3.

BACKGROUND: Gamma irradiation of blood products is used to prevent transfusion-associated graft-versus-host disease by inhibiting the proliferation of lymphocytes that are implicated in the disease. Gamma irradiation also damages the red blood cells (RBCs). It is unknown whether hypoxia reduces the efficacy of gamma irradiation in inhibiting lymphocyte proliferation (LP). The objectives of the study were to investigate the effects of hypoxia on gamma irradiation-induced inhibition of LP and on the in vitro properties of RBCs. MATERIALS AND METHODS: Forty-four units (300-340 ml each) of less than 8-h-old ABO-matched leukocyte reduced red cell concentrates (LR-RCC) in additive solution 3 were pooled in pairs. Peripheral blood mononuclear cells were isolated from non-leukocyte reduced RCCs and added back to the pool at a final concentration of 2 × 105 /ml. The pool was divided equally into a conventional storage bag A and a hypoxic processing and storage bag B. The units were gamma-irradiated at 25Gy on day 7 for the LP experiment and on either day 7 or 14 for the RBC quality experiments. LP was measured using a limiting dilution assay, and several in vitro metrics of RBCs were measured. RESULTS: Gamma irradiation inhibited T-lymphocyte proliferation by 4.7 × 104 -fold reduction in both hypoxic and conventional storage. The in vitro metrics of RBC quality were better preserved in hypoxic storage. DISCUSSION: T lymphocytes present in hypoxic RBC are equally susceptible to gamma irradiation as conventional storage. Hypoxic storage also reduces the deleterious effects of gamma irradiation on RBCs.