Red Cell Recovery Research Articles

Seven-years post allogeneic hematopoietic stem cell transplantation pure red cell aplastic anemia cured with daratumumab: A case report and review of literature

BACKGROUND Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is currently the only viable method of curing patients with acute myeloid leukaemia. In 30% to 50% of patients, donors and recipients have some level of ABO blood group incompatibility. ABO blood group incompatibility can cause antibodies against the donor's red blood cells to persist in the recipient's body, resulting in a delay of several months in the recovery of red blood cells. A number of different treatments have been reported for post-transplant pure red cell aplastic anaemia (PRCA), such as plasmapheresis, donor lymphocyte infusions, anti-thymocyte globulin, rituximab and steroids. CASE SUMMARY A 41-year-old female diagnosed with acute myeloid leukaemia underwent peripheral blood allogeneic haematopoietic stem cell transplantation in November 2013 from an HLA matched unrelated donor. The donor was AB-positive and the recipient was O-positive. The patient was diagnosed with PRCA three months after receiving the donor stem cell transplant. After failing multiple lines of therapy, the patient applied for daratumumab. After receiving three doses of daratumumab, the patient developed a reticulocyte response and no longer required blood transfusions. CONCLUSION The use of daratumumab anti-CD38 for the remove of plasma cells is safe and effective and may be tried for refractory patients with PRCA after undergoing allo-HSCT for ABO incompatibility.

MED16 Negatively Regulates Erythropoiesis and Myelopoiesis through Modulation of Chromatin Accessibility

Tissue-specific transcription factors work in conjunction with numerous coregulators to modulate transcription networks during hematopoiesis; disruptions in this process may lead to hematopoietic disorders, including myelodysplastic syndromes (MDS). While the Mediator complex is known to regulate various aspects of hematopoiesis, the mechanisms underlying subunit-specific functions have not been fully understood. Here, we uncovered that MED16, a core component of Mediator tail module, serves as a crucial negative regulator of erythropoiesis and myelopoiesis by controlling chromatin accessibility at specific target genes. To investigate the function of MED16 in vivo, we transplanted control (shSCR) or MED16-deficient (shMED16) human cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) into immunodeficient mice. Mice receiving shMED16 HSPCs showed a higher proportion of human erythroid and myeloid cells in the bone marrow (BM), while B cell and T cell reconstitution remained unaffected. MED16 deficiency led to an increased number of myeloid colonies and a more pronounced increase in erythroid colonies in the colony-forming assay. During erythroid differentiation, MED16 deficiency resulted in an elevated ratio of CD235a+ cells and increased enucleation. Furthermore, hematopoietic-specific Med16 knockout ( Mx1-Cre × Med16 -/- Med16 KO)mice showed normal BM and spleen cellularity, with an increased frequency of common myeloid progenitors (CMPs). Med16 KO mice exhibited a higher frequency of CD11b+ myeloid cells and Ter119+ erythroid cells in the BM and spleen. A higher percentage of enucleated erythrocytes were observed in the BM of Med16 KO mice. Additionally, in the model of phenylhydrazine-induced hemolytic anemia, Med16 KO mice showed accelerated red blood cell recovery and pronounced splenomegaly, highlighting the crucial role of Med16 in stress erythropoiesis. Our findings demonstrate that MED16 plays a repressive role in hematopoietic development. To elucidate how MED16 regulates gene expression, we conducted single-cell RNA-seq and ATAC-seq of cultured CD34+ cells after MED16 knockdown in primary human CD34+ HSPCs. MED16 deficiency resulted in upregulation of erythroid and innate immunity genes in erythroid and myeloid cells, respectively. Interestingly, MED16 deficiency led to significantly increased chromatin accessibility around the transcription start sites of upregulated genes. Considering the Mediator complex often interacts with transcription factors to convey information, we conducted MED16 chromatin IP sequencing (ChIP-seq). The majority of MED16 occupancy peaks were located at promoters (59.68%). Colocalization analysis of MED16 with chromatin occupancy of hematopoietic-related transcription factors revealed significant overlap with RUNX1. To unveil the mechanism of MED16-mediated transcriptional repression, we conducted immunoprecipitation coupled with mass spectrometry on cultured CD34+ cells to identify MED16-interacting nuclear proteins. We identified the FAcilitates Chromatin Transcription (FACT) complex as a potential candidate. ChIP-seq of the FACT complex subunit SPT16 showed weakened chromatin occupancy signals upon MED16 deficiency. Similar results were observed in mouse BM Ter119+ erythroblasts. These findings indicate that MED16 plays a crucial role in modulating chromatin accessibility and transcriptional repression, potentially through cooperation with RUNX1 and the FACT complex. Furthermore, we found that MED16 overexpression impeded erythroid cell enucleation, similar to MDS erythroid dysplasia. We observed higher MED16 expression in BM-derived CD34+ cells from 183 MDS patients across different subtypes compared to healthy individuals. Interestingly, knocking down MED16 in bone marrow mononuclear cells from MDS patients restored normal erythropoiesis. Transcriptome analysis revealed that MED16 deficiency upregulated innate immunity and erythroid genes. Together, these findings shed light on the previously unknown role of MED16 in hematopoiesis and its association with MDS. Our study provides mechanistic insights into the lineage commitment and developmental progression of hematopoiesis governed by Mediator and may have implications for the treatment of hematological disorders.

M6A Mediated Ribostasis of RNA Stress Granule Assembly Governs Blood Development and Regeneration

Hematopoietic stem cell (HSC) form diverse cell types and requires transcription and translation regulation. Using CD34+ human hematopoietic stem and progenitor cells (hCD34+) combined with N6-methyladenosine (m6A) sequencing, we identified a novel regulatory mechanism via stress granule regulation during erythropoiesis. Our single-nucleotide resolution m6A analysis of the whole transcriptome identified ~4800 transcripts to be m6A modified and showed demethylation (>2-14 fold) during erythropoiesis. Genome-wide CHIP-seq analysis of hCD34+ cell erythropoiesis with anti-Gata1 showed an enhancer enrichment at the ALKBH5 RNA demethylase gene. Using ALKBH5 CRISPR Cas9 KO strategy combined with hCD34+ cell in-vitro differentiation protocol we observed a block in hCD34+ cell erythropoiesis thus validating Alkbh5 role in erythropoiesis. hCD34+ ALKBH5 KO cells showed a failure of RNA m6A demethylation and discovered stress granule (SGs) transcripts m6A levels to be upregulated (>2-4 fold, p=0.01). Stress granules are membrane-less organelles composed of various RNA binding proteins. SGs form a hub in the cytoplasm that governs RNA metabolism and global proteome by translational regulation. Our analysis showed ATXN2, G3BP1/2, Tia-1,and PABPC1 SGs transcripts are m6a modified at 3'UTR. K562 cell ALKBH5 KO erythropoiesis model led to massive m6A upregulation on ATXN2 3'UTR (>14 fold, p=0.001) and other SGs transcripts. Mass spectrometry analysis of hCD34+ ALKBH5 KO cell proteome showed downregulation of ATXN2(>2 fold, -log P value2), confirming 3' UTR m6A mediated protein synthesis regulation. ALKBH5 KO hCD34+ cells showed aggregation of SGs protein in the cytoplasm due to altered SG protein stoichiometry(>4-10 fold, p=0.001). Zebrafish alkbh5 KO showed defective blood development and anemia. STED microscopy of the whole kidney marrow of alkbh5 KO fish showed the presence of G3BP and ATXN2 SGs with m6A labeled RNAs. alkbh5 KO zebrafish blood and kidney marrow analysis also showed increased myeloid and lymphoid lineages indicating early lineage bias. We used the alkbh5 KO zebrafish model and subjected it to phenyl hydrazine-induced anemia to study its role in stress-induced regeneration. Using FACS analysis of whole kidney marrow, we show red cell recovery failure in alkbh5 KO zebrafish models. Temporal analysis of alkbh5 KO marrow cells during PHZ-induced acute anemia and regeneration showed a systemic failure to resolve SGs compared to controls. We confirmed these results in the mouse ALKBH5 KO model establishing the role of ALKBH5 in stress erythropoiesis. Furthermore, hCD34+ ATXN2 KO combined with Methocult-colony formation assay showed defects in erythroid and myeloid lineages. Super-resolution microscopy of hCD34+ ATXN2 KO cells phenocopied the presence of disease-associated ALS (amyotrophic lateral sclerosis) stress granules with TDP-43, G3BP1/2, PABPC1, and Tia-1 enriched with m6A labeled RNAs. Using the K562 ALKBH5 KO erythropoiesis model and ATXN2 lentiviral overexpression, we effectively resolved stress granules and blocks in erythropoiesis. In summary, m6A marks on ATXN2 mRNA regulate SG biology, affecting HSC differentiation and impacting blood development. SG-mediated regulation of HSC fate may highlight new therapeutic strategies for blood disorders.

Alternative Bone Marrow Transplantation with Post-Transplant Cyclophosphamide (PTCy) As Initial Therapy for Acquired Severe Aplastic Anemia (SAA)

Immunosuppressive therapy (IST) is standard front-line treatment for SAA, except for patients aged <20 years with a suitable HLA-matched sibling donor (MSD) for BMT. The hematopoietic response after IST, even with the addition of eltrombopag, is ~ 70%-80% and 5-year survival is 60% to 85%. Failure-free survival (alive and in remission without clonal disease) beyond 10 years after IST is ~50%. In contrast, long-term survival after BMT is ~90% in patients aged < 20 years, and 75% in older patients. Currently, BMT with an unrelated or HLA-haploidentical (haplo) donor is reserved for failure of IST because of morbidity and mortality concerns. PTCy improves the safety and efficacy of haplo BMT by facilitating engraftment and decreasing graft-vs-host disease (GVHD), with survival comparable to MSD BMT. Here we review the outcomes of alternative donor BMT and PTCy in treatment-naïve (TN) SAA patients. Eligible patients met criteria for SAA and were TN. Fanconi anemia and telomere biology disorder patients were excluded. Non-myeloablative (NM) conditioning included rabbit antithymocyte globulin (0.5 mg/kg day-9 and 2 mg/kg on days -8,-7), fludarabine (30mg/m2/day on days -6 to -2), low dose CY (14.5 mg/kg on days -6, -5) and total body irradiation (TBI) (200cGy or 400 cGy day -1). After the marrow graft was infused on day 0, cyclophosphamide 50 mg/kg/day IV was administered on days 3 and 4 post-transplant. All received mycophenolate mofetil (MMF) from day 5-35 and tacrolimus from day 5 through 1 year. Neutrophil recovery was defined as occurring on the first of three consecutive days of an ANC >1.0 x109/L. Red cell recovery was defined as days from transplant to the last red blood cell transfusion. Platelet recovery was defined as the first day of a platelet count >50,000 without transfusion in the preceding 7 days. Thirty-two eligible patients were treated at Johns Hopkins between 9/2016 and 5/2022. Median follow-up for all patients is 40 months (range 2-78). Table 1 shows demographics. The median age was 25 years (range 3-63) with 24 patients age >20 years, and 56% were males. Median time to neutrophil recovery was 20 days (range 14-88). Median time to red cell recovery was 25 days with 90% transfusion independence by day 60. Median time to platelet recovery was 25 days with 90% transfusion independence by day 100. TBI dose was increased from 200 to 400 cGy to reduce graft failure after graft loss in 3 of the first 7 patients; 2 of these 3 died (from infection and graft failure) and one has been re-transplanted and is now 100% donor. Overall survival at 3 years post-transplant is 93%. (Figure 1) All 24 consecutive patients who received 400 cGy are alive and well with engraftment. CMV reactivated in 13 patients, and 6 required therapy. Rates of acute and chronic GVHD were both 7%, none developed grade 3-4 acute or extensive chronic GVHD, and all patients beyond day 365 are off treatment for GVHD. No secondary malignancies have been observed. NM BMT with PTCy, using mostly haplo donors, can cure SAA. Increasing TBI dose from 200 to 400 cGy improves engraftment. Rates of infection and mortality are not increased compared to standard upfront IST. Historical data show that >50% of patients in this age range require transplantation subsequent to IST. Upfront BMT, as described here, allows patients to come off immunosuppression by 6-12 months post-BMT, prevents the morbidity of prolonged IST, and may reduce healthcare costs. Most patients receiving 200 cGy TBI maintain fertility; we are monitoring fertility rates after 400cGy. This approach forms the basis for the upcoming Bone Marrow Transplant Clinical Trials Network multicenter study in TN acquired SAA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Sec14l4 SNP Mice Have Increased Susceptibility to Oxidative Hemolysis through Modulation of G6PD and the Pentose Phosphate Pathway

Red blood cell function and recovery following transfusion is influenced by a variety of factors including sex, race, sickle cell trait and single nucleotide polymorphisms (SNPs). In collaboration with the REDS III program, we performed a genome wide association study identifying SNPs that are associated with in vitro osmotic, oxidative and spontaneous storage hemolysis stress. Sec14L4, was one of 27 genes identified with enhanced hemolysis, specifically following oxidative insult and later confirmed by an independent study to be associated with decreased hemoglobin increments following transfusion. SEC14L4 is an understudied, highly conserved phosphatidylinositol transfer protein that is predicted to reside with the Golgi Apparatus. While the function of SEC14L4 is not understood, it is clear that polymorphisms in this gene have clinical impact and understanding its role in red cell biology and transfusion is critical. We generated a novel humanized Sec14L4 SNP and knockout mouse model to assess RBC metabolism, function and transfusion outcomes following storage. While Sec14l4 KO mice have complete loss of SEC14L4 protein expression, the SNP has an approximate 50% decrease in expression, suggesting the mutation decreases protein and/or mRNA stability. Interestingly, knockout mice experience anemia with decreased hemoglobin levels and lower hematocrit at baseline. Following 12 days of storage (equivalent to 42 days of human blood storage) Sec14l4 knockout RBCs have increased hemolysis and are more susceptible to lysis following treatment with the oxidant diamide. Tracing experiments with 1,2,313C3-glucose from Sec14l4 SNP mouse RBCs reveals decreased levels of glucose-6-phosaphate dehydrogenase (G6PD) and depression of metabolic fluxes through the pentose phosphate pathway with lower levels of 13C3-6-phosphogluconate and nicotinamide adenine dinucleotide phosphate (NADP+), and reduced ADP and ATP. Compensatory increases in glycolytic flux was also observed. G6PD is the rate liming enzyme of the pentose phosphate pathway which is the sole producer of NADPH in the red blood cell. NADPH is an essential cofactor for glutathione reductase and peroxidase, the primary erythrocyte antioxidant enzymes. Red blood cells deficient for G6PD undergo acute hemolysis if exposed to oxidative stress due to decreased antioxidant capacity. These data suggest that red blood cells deficient in SEC14L4 phenocopy G6PD deficient red cells, possibly explaining increased susceptibility to oxidative insult. Lipidomic evaluation also reveled increased accumulation of oxidized lipids during storage (12-HETE and 15-HETE). Oxilipins are markers of poor post-transfusion recovery in stored mouse and human red blood cells. Together these data indicate that SEC14L4 is an important player in red blood cell stability during storage, oxidative stress, and following transfusion. How a Golgi resident protein influences the adult red cell is not yet understood but suggests a role for SEC14L4 in early erythrocyte development which has an effect over the lifetime of the cell, with the result of increased stability and antioxidant capacity. In support of this hypothesis, Sec14l4 is highly expressed in the bone marrow with decreasing expression as the red cell terminally differentiates. Future work will confirm proteomic, metabolomic and lipidomic findings and focus on how SEC14L4 may be regulating the G6PD pathway in mature red cells.

A randomized trial of blood donor iron repletion on red cell quality for transfusion and donor cognition and well-being

AbstractAlthough altruistic regular blood donors are vital for the blood supply, many become iron deficient from donation-induced iron loss. The effects of blood donation–induced iron deficiency on red cell transfusion quality or donor cognition are unknown. In this double-blind, randomized trial, adult iron-deficient blood donors (n = 79; ferritin < 15 μg/L and zinc protoporphyrin >60 μMol/mol heme) who met donation qualifications were enrolled. A first standard blood donation was followed by the gold-standard measure for red cell storage quality: a 51-chromium posttransfusion red cell recovery study. Donors were then randomized to intravenous iron repletion (1 g low-molecular-weight iron dextran) or placebo. A second donation ∼5 months later was followed by another recovery study. Primary outcome was the within-subject change in posttransfusion recovery. The primary outcome measure of an ancillary study reported here was the National Institutes of Health Toolbox–derived uncorrected standard Cognition Fluid Composite Score. Overall, 983 donors were screened; 110 were iron-deficient, and of these, 39 were randomized to iron repletion and 40 to placebo. Red cell storage quality was unchanged by iron repletion: mean change in posttransfusion recovery was 1.6% (95% confidence interval −0.5 to 3.8) and −0.4% (−2.0 to 1.2) with and without iron, respectively. Iron repletion did not affect any cognition or well-being measures. These data provide evidence that current criteria for blood donation preserve red cell transfusion quality for the recipient and protect adult donors from measurable effects of blood donation–induced iron deficiency on cognition. This trial was registered at www.clinicaltrials.gov as NCT02889133 and NCT02990559.

Small-molecule fulvic acid with strong hydration ability for non-vitreous cellular cryopreservation

SummaryThe exploitation of biocompatible ice-control materials especially the small molecules for non-vitreous cryopreservation remains challenging. Here, we report a small molecule of fulvic acid (FA) with strong hydration ability, which enables non-vitreous cellular cryopreservation by reducing ice growth during freezing and reducing ice recrystallization/promoting ice melting during thawing. Without adding any other cryoprotectants, FA can enhance the recovery of sheep red blood cells (RBCs) by three times as compared with a commercial cryoprotectant (hydroxyethyl starch) under a stringent test condition. Investigation of water mobility reveals that the ice-control properties of FA can be ascribed to its strong bondage to water molecules. Furthermore, we found that FA can be absorbed by RBCs and mainly locates on membranes, suggesting the possible contribution of FA to cell protection through stabilizing membranes. This work bespeaks a bright future for small-molecule cryoprotectants in non-vitreous cryopreservation application.

Cryopreservation of rare pediatric red blood cells for support following bone marrow transplant.

A 2-year-old, 10.8kg male pediatric patient with X-linked chronic granulomatous disease (CGD) with McLeod syndrome (MLS) was scheduled for a hematopoietic stem cell transplant (HSCT). Identification of allogenic red blood cells (RBC) for post-transplant support was unsuccessful prompting the development of a customized method to collect and freeze rare autologous pediatric cells. A protocol was developed for the collection of small volume pediatric whole blood (WB) via peripheral venipuncture with collection into 10ml syringes containing anticoagulants. Additionally, a closed system RBC glycerolization and deglycerolization instrument was adapted to process small volume, non-leukoreduced WB. Both collection and WB processes were validated. In total 4 approximately 100 ml autologous units were collected and frozen. Two units were thawed, deglycerolized, and used for clinical transfusion support. To appreciate processing impacts on RBC rigidity, ektacytometry was performed on pre-processed and post-deglycerolization samples. Free hemoglobin (HGB) of validation units after thawing/deglycerolization was <150 mg/dL with an average red cell recovery of 85%. These units also showed little difference between pre-and post-processing Lorrca deformability curves or membrane rigidity. Two pediatric units were thawed and deglycerolized for transfusion. Free HGB was 70 mg/dL and 50 mg/dL post-thaw, and these RBCs had a slight decrease in deformability and increased membrane rigidity. Customized WB collection, glycerolization, freezing, and deglycerolization processes were developed to successfully support a pediatric patient with CGD and MLS after autologous HSCT. Both pediatric units showed increased membrane rigidity post-deglycerolization which may be a consequence of the CGD and MLS genetic background.

Sex-specific genetic modifiers identified susceptibility of cold stored red blood cells to osmotic hemolysis

BackgroundGenetic variants have been found to influence red blood cell (RBC) susceptibility to hemolytic stress and affect transfusion outcomes and the severity of blood diseases. Males have a higher susceptibility to hemolysis than females, but little is known about the genetic mechanism contributing to the difference.ResultsTo investigate the sex differences in RBC susceptibility to hemolysis, we conducted a sex-stratified genome-wide association study and a genome-wide gene-by-sex interaction scan in a multi-ethnic dataset with 12,231 blood donors who have in vitro osmotic hemolysis measurements during routine blood storage. The estimated SNP-based heritability for osmotic hemolysis was found to be significantly higher in males than in females (0.46 vs. 0.41). We identified SNPs associated with sex-specific susceptibility to osmotic hemolysis in five loci (SPTA1, KCNA6, SLC4A1, SUMO1P1, and PAX8) that impact RBC function and hemolysis.ConclusionOur study established a best practice to identify sex-specific genetic modifiers for sexually dimorphic traits in datasets with mixed ancestries, providing evidence of different genetic regulations of RBC susceptibility to hemolysis between sexes. These and other variants may help explain observed sex differences in the severity of hemolytic diseases, such as sickle cell and malaria, as well as the viability of red cell storage and recovery.

Recovery of platelet-rich red blood cells and acquisition of convalescent plasma with a novel gravity-driven blood separation device.

ObjectivesOur objectives were to determine the separation characteristics and blood product quality of a gravity‐driven microfiltration blood separation system (HemoClear, The Netherlands).BackgroundA range of centrifugal blood separation devices, including intraoperative cell salvage devices (cell savers) and apheresis machines, are available to assist in preparing both allogenic and autologous blood products. These devices are expensive to operate and require extensive training.Methods and MaterialsNine whole blood units were collected under standard conditions and analysed for haematological parameters, thromboelastographic properties, platelet morphology and activation, and red blood cell (RBC) deformability and morphology. Three whole blood units were separated by means of the HemoClear device, into a liquid and cellular component. The cellular component was diluted with SAGM and cold stored for 14 days. To simulate cell salvage six whole blood units were diluted with isotonic saline, followed by multiple HemoClear separation rounds.ResultsThe recovery of both RBCs (100 ± 1.6%) and white blood cells (99 ± 4.5%) after undiluted filtration were very high, while platelet recovery was high (83 ± 3.0%). During the filtration, and cold storage after filtration storage both the non‐deformable RBC fraction and the RBC maximum elongation remained stable. Parameters of thromboelastography indicated that platelets remain functional after filtration and after 7 days of cold storage. In the cell salvage simulation the total protein load in the cellular fraction was reduced by 65 ± 4.1% after one washing round and 84 ± 1.9% after two consecutive washing rounds.ConclusionThe novel blood filter studied effectively separates whole blood into diluted plasma and platelet‐rich RBCs. Moreover, the device effectively washed diluted whole blood, driving over 80% of proteins to the liquid component.

Effect of radioiodine therapy (131I) on blood cells of differentiated thyroid carcinoma patients

Thyroid cancer accounts for about 1% of all types of cancers, with about 80% of cases being differentiated and 20% undifferentiated. Our study was conducted on 209 Differentiated Thyroid Carcinoma (DTC) patients treated at the Department of Nuclear Medicine of Nghe An Cancer Hospital with 91.39% of patients having papillary thyroid carcinoma, with the mean age of 47.91 ± 12.08. The geographic distribution of these patients was determined to be 68.42% coastal plains. Patients living in mountainous areas accounted for only 6.22%. For our experiment, treatment for patients including having the tumor removed and thyroid tissue destroyed using 131I. Patients were then prescribed with alternative hormones varying at different doses depending on the degree and stage of cancer. Among 209 patients, 8 patients were treated with 30mCi dose(3.83%); 196 patients treated with the dose of 100–150 mCi (93.78%) and 5 patients treated 200–250 mCi (2.39%). After 3–5 days of treatment, we observed significant changes in blood count with p&lt;0.05. Initially, erythrocytes and hemoglobin count decreased slightly for 30–150 mCi dose patients but with 200–250 mCi dose patients, those indexes fell below the reference level. The platelet index decreased significantly after 3–5 days of treatment (p&lt;0.05). Total leukocytes, lymphocytes and mono count decreased sharply after treatment (p&lt;0.05) but neutrophils changed very little. After 3–6 months of treatment, the blood cell formula gradually stabilized, the recovery of red blood cells took place faster than the white blood cells and platelets. The recovery of blood cells of patients treated with doses of 200–500 mCi took place more slowly (p&lt;0.05). Thus, the use of 131I in the treatment of DTC is a common and effective method, but they may also be at risk of impairing blood cells, especially in high-dose 131I patients. However, this change is still within the allowable limits and after 6 months of treatment, the blood cell counts tend to return to their original values. The speed of recovery depends on the individual blood cell line and the amount of treatment. This research result can be used as a reference for deciding on 131I dosage and supportive measures, interventions in treatment, helping patients avoiding the decrease of blood cells and increase the effectiveness of the treatment process.

Methods of pathogen inactivation in whole blood and red blood cells: current state of knowledge

Although pathogen reduction technology was implemented for platelet concentrates and plasma, the risk of pathogen transmission has not been completely eliminated as no inactivation procedures were implemented for red blood cells and whole blood. Research was therefore focused on developing methods for effective pathogen inactivation in red blood cell components. Attempts were made to apply either chemical compounds (porphyrins and Sylsense compounds) or photosensitizers such as methylene blue (Theraflex MB Plasma System) and amotosalen hydrochloride (Intercept System) already in use for pathogen inactivation in plasma. None proved effective for pathogen inactivation in red blood cells. Approval was recently given to pathogen inactivation methods based on S-303 compound (for red blood cells) and with riboflavin (for whole blood). Clinical trials are ongoing. Pilot studies have shown that during storage of packed red blood cells subjected to pathogen inactivation with S-303 demonstrated slight loss of red blood cells, decrease in hemoglobin concentration, significantly lower lactate concentration, and lower pH. Pathogen inactivated whole blood stored at room temperature for up to seven days showed slight hemolysis (within the normal range). This paper presents several pilot clinical trials with pathogen inactivated red blood cells or whole blood. It focuses primarily on the recovery of red blood cells in the recipient's organism and on hemoglobin concentration.

Effects of captopril against radiation injuries in the Göttingen minipig model of hematopoietic-acute radiation syndrome

Our laboratory has demonstrated that captopril, an angiotensin converting enzyme inhibitor, mitigates hematopoietic injury following total body irradiation in mice. Improved survival in mice is correlated with improved recovery of mature blood cells and bone marrow, reduction of radiation-induced inflammation, and suppression of radiation coagulopathy. Here we investigated the effects of captopril treatment against radiation injuries in the Göttingen mini pig model of Hematopoietic-Acute Radiation Syndrome (H-ARS). Minipigs were given captopril orally (0.96 mg/kg) twice daily for 12 days following total body irradiation (60Co 1.79 Gy, 0.42–0.48 Gy/min). Blood was drawn over a time course following irradiation, and tissue samples were collected at euthanasia (32–35 days post-irradiation). We observed improved survival with captopril treatment, with survival rates of 62.5% in vehicle treated and 87.5% in captopril treated group. Additionally, captopril significantly improved recovery of peripheral blood mononuclear cells, and a trend toward improvement in recovery of red blood cells and platelets. Captopril significantly reduced radiation-induced expression of cytokines erythropoietin and granulocyte-macrophage colony-stimulating factor and suppressed radiation-induced acute-phase inflammatory response cytokine serum amyloid protein A. Using quantitative-RT-PCR to monitor bone marrow recovery, we observed significant suppression of radiation-induced expression of redox stress genes and improved hematopoietic cytokine expression. Our findings suggest that captopril activities in the Göttingen minipig model of hematopoietic-acute radiation syndrome reflect findings in the murine model.

An explicit correlation between surface functionality, wettability, and leukocyte removal by electrospun filter media

The major motive of this investigation was to derive a correlation between surface functionality, wettability, and leukodepletion efficiency of electrospun filter media. Towards this, different extents of hydrophilicity and various functionalities were introduced to the electrospun poly (ethylene-co-vinyl alcohol) (EVAL) filter media via photografting of 2-hydroxyethyl acrylate (HEA) branches, photografting of N-(3-sulfopropyl)-N-methacroyloxyethyl-N,N- dimethylammonium betaine (SMDB) branches, and integration of glycine. All the filter media were designed to asymmetric filters with fiber sizes varying from 1.7 ± 0.2 μm to 2.4 ± 0.2 μm, pore sizes from 7.8 ± 1.5 μm to 23.3 ± 5.0 μm, water contact angles from 69 ± 10° to 134 ± 3 critical wetting surface tension (CWST) from 61.8 mN/m to > 88 mN/m and their performance was ascertained by carrying out whole blood filtration studies. It was observed that hydrophilic filter media resulted in a decreased adhesion of white blood cells (WBCs) than the unmodified EVAL filter media due the formation of a hydration layer. However, the hydrophilic SMDB grafted EVAL (PSMBD-g- EVAL) showed a high WBC adhesion than expected because of the net negative polarity of the PSMDB branches by which they could grasp the WBC even in presence of hydration layer. The efficacy of WBC adhesion by hydrophobic glycinated EVAL (EVAL-GLY) filter media was nearly as that of unmodified EVAL while the red blood cell (RBC) recovery was less pronounced (67 % lower than unmodified EVAL) due to the pore occlusion. However, there was no significant change in platelet adhesion for all filter media except PHEA-g-EVAL. Hemolysis induced by all the modified filter media was notably lesser than that of unmodified EVAL and was nearly zero. Thus, it is concluded that WBC and platelet adhesion are interdependent under dynamic conditions and it is difficult to achieve a selective adhesion of one in presence of the other.

Clodronate-liposomes aggravate irradiation-induced myelosuppression by promoting myeloid differentiation

Purpose Clodronate-liposomes (Clod-Lip) is an effective candidate drug for treating chronic myelomonocytic leukemia, autoimmune hemolytic anemia and immune thrombocytopenic purpura in mice experiments. But its role in hematopoietic recovery after acute myelosuppression is still unknown. We aim to explore the function and underlining mechanisms of Clod-Lip on hematopoietic reconstitution after sublethal dose irradiation in mice. Materials and methods Mice at 8-10 weeks received a total-body sublethal dose γ-irradiation (TBI) and injected with Clod-Lip or PBS-Liposomes (PBS-Lip) every 4 days after TBI. The survival rate of each group was recorded. Flow cytometry was used to analyze changes in hematopoietic stem cells and their progenies in bone marrow. ELISA and RT-qPCR were used for the analysis of hematopoietic regulatory factors. Regarding IL-1β inhibition, 25 mg/kg diacerein or an equal volume of DMSO was intraperitoneally injected into mice every day after TBI. Results In sublethal dose-irradiated mice, Clod-Lip reduced the survival rate, the total number of bone marrow and hematopoietic stem cells, delayed peripheral blood recovery of red blood cells and platelets. However, it could increase the number of CMP, MEP and myeloid cells, which suggested that Clod-Lip could induce HSC to myeloid differentiation in vivo. We further verified that Clod-Lip may induce myeloid differentiation by bone marrow microenvironmental factor IL-1β. Conclusions In summary, this study suggested that Clod-Lip may aggravate inhibitor effect of hematopoietic function and promote myeloid differentiation in myelosuppression mice model.

Purification of Plasmodium and Babesia- infected erythrocytes using a non-woven fabric filter.

The purification of parasite-infected erythrocytes from whole blood containing leucocytes is crucial for many downstream genetic and molecular assays in parasitology. Current methodologies to achieve this are often costly and time consuming. Here, we demonstrate the successful application of a cheap and simple Non-Woven Fabric (NWF) filter for the purification of parasitized red blood cells from whole blood. NWF filtration was applied to the malaria-parasitized blood of three strains of mice, and one strain of rat, and to Babesia gibsoni parasitized dog blood. Before and after filtration, the white blood cell (WBC) removal rates and red blood cell (RBC) recovery rates were measured. After NWF filter treatment of rodent malaria-infected blood, the WBC removal rates and RBC recovery rates were, for Kunming mice: 99.51%±0.30% and 86.12%±8.37%; for BALB/C mice: 99.61%±0.15% and 80.74%±7.11%; for C57 mice: 99.71%±0.12% and 84.87%±3.83%; for Sprague-Dawley rats: 99.93%±0.03% and 83.30%±2.96%. Microscopy showed WBCs were efficiently removed from infected dog blood samples, and there was no obvious morphological change of B. gibsoni parasites. NWF filters efficiently remove leukocytes from malaria parasite-infected mouse and rat blood, and are also suitable for filtration of B. gibsoni-infected dog blood.

Abo不相容性和抗a / B异凝集素滴度对血液恶性肿瘤清髓处理后输血需求和无关脐带血移植的早期结果的影响

Abstract Background:ABO incompatibility is not considered a main contraindication to allogeneic hematopoietic stem cell transplantation (aHSCT). However, it has been associated with a number of immunohematological complications. The effects of ABO incompatibility on aHSCT remain controversial.The change of isoagglutinin titers and early clinical outcomes were analyzed after unrelated cord blood transplantation (UCBT) with ABO-incompatibility donor. Methods:252 patients with hematological malignant diseases and other hematological disorders who underwent unrelated UCBT from January 2019 to April 2020 were retrospectively analyzed in this research. Patients were studied in identical, major, minor and bidirectional mismatch groups. Immunoglobulin m (IgM) isoagglutinin titers were tested one day before the transplant (-1 day), 2 weeks post-transplant, 4 weeks post-transplant and 6 weeks post-transplant. R esults:76 match,71 major mismatch, 70 minor mismatch and 35 bidirectional unrelated UCBT were identified. The median neutrophil, PLT and red blood cell (RBC) recovery days were 18, 38 and 22, respectively. ABO mismatch did not influence the neutrophil, PLT and RBC engraftment. The median of RBC transfusion in 30 days were 5 units and PLT were 6 units. There were no statisitcal difference in 0-30 days RBC and PLT transfusion after UCBT. 31-100 days transfusion was similar to in 30 days transfusion. No patients developed pure red cell anemia (PRCA). -1day IgM titers ≥1:16 did not develop higher risk of grade II-IV aGVHD when compared with titers≤1:8 group. However, we detected a marginal higher PLT transfusion in 30 days after transplant at antibody titers ≥1:16 group when compared with titers≤1:8 (P=0.051). In the major and bidirectional groups, we found that group O IgM anti-donor antibodies were displayed a significant higher than the group B anti-A titer (p&amp;lt;0.001) in setting the time one day before the transplant, but no significant with group A. 2 weeks after the transplant, group B anti-A was still showed significant lower than the group O anti-A (p&amp;lt;0.001). 4 weeks after the UCBT, we observed a modest, but no statistical significant lower titers of group B anti-A antibodies as compared with O group (P=0.097). 6 weeks after the transplant, there were no statistical significant among group O, A and B. In the multivariable Cox regression model, transfusion of ≥5 RBC units in 30 days after UCBT (HR=1.727, 95%CI=1.020-2.926, P=0.042) and PLT engraftment ≥38 days (HR=1.964, 95%CI=1.134-3.401, P=0.016) were correlated to greater risk of grade severe aGVHD. Conclusion:This study showed that ABO mismatch did not influence the neutrophil, PLT and RBC recovery time.Group O IgM anti-donor isoagglutinins in recepients showed a higher titers than the group B in setting with the time (-1 days pre-transplant, 2 weeks post-transplant, 4 weeks post-transpant). Pre-transplant higher anti-donor isoagglutinins were associated with more PLT transfusion requirements after UCBT. More RBC transfusion (≥5 units) and longer PLT recovery time (≥38 days) showed a higher incidence of severe aGVHD. Disclosures No relevant conflicts of interest to declare.

Astragalus polysaccharide has a protective effect on hematopoiesis in an irradiated mouse model and decreases apoptosis in megakaryocytes.

Huangqi, the dried root of Radix Astragali, is an essential herb in Traditional Chinese Medicine and has been used to promote hematopoiesis for centuries. Astragalus polysaccharide(ASPS), the bioactive compound of Huangqi, serves a crucial role in hematopoiesis. The aim of the present study was to investigate the hematopoietic effects, in particular the thrombopoietic effects, and the molecular mechanisms of ASPS using an irradiation‑induced myelosuppressive mouse model. Colony‑forming unit assays, flow cytometric analysis of apoptosis, ELISAs, Giemsa staining and western blotting were performed to determine the hematopoietic and anti‑apoptotic effects of ASPS. The results demonstrated that ASPS enhanced the recovery of red blood cells at day21 following treatment, as well as platelets and white blood cells at day14. In addition, ASPS promoted colony formation in all lineages (megakaryocytes, granulocyte monocytes, erythroid cells and fibroblasts). The morphological study of the bone marrow demonstrated that tri‑lineage hematopoiesis was preserved in the ASPS‑ and thrombopoietin(TPO)‑treated groups compared with the control group. The overall cellularity (mean total cell count/area) of the ASPS‑treated group was similar to that of the TPO‑treated group. Additionally, <em>invitro</em> experiments indicated that treatment with 100<em>µ</em>g/ml ASPS exhibited the maximum effect on colony formation. ASPS attenuated cell apoptosis in megakaryocytic cells via inhibiting the mitochondrial caspase‑3 signaling pathway. In conclusion, ASPS promoted hematopoiesis in irradiated myelosuppressive mice possibly via enhancing hematopoietic stem/progenitor cell proliferation and inhibiting megakaryocytes apoptosis.

Investigation of the potency of leukodepletion filter membranes immobilized with bovine serum albumin via polydopamine spacer

Conjugation of biomolecules over membrane surfaces intended for blood contacting applications has been proven worthwhile for the improvement of their hemocompatibility. In conjunction with this factuality, this manuscript for the first time unveils the feasibility of bovine serum albumin (BSA) immobilization via polydopamine precursors on electrospun poly(ethylene-co-vinyl alcohol) (EVAL) leukodepletion filter membranes. The successful immobilization of different extents of BSA on EVAL membranes was endorsed by the Attenuated Total Reflectance—Fourier Transform Infrared (ATR-FTIR) Spectrum, variations in the wettability, electron microscopic images revealing the alterations in the fiber diameter and porousness of membranes. An interesting backlash was encountered in the in vitro plasma protein adsorption and in vitro platelet adhesion on the BSA-immobilized membranes as they exhibited a higher protein adsorption and lower platelet adhesion in comparison with neat EVAL. The infiltration of immobilized BSA to the leukodepletion capacity of those membranes was preliminarily studied by in vitro blood-material interactions followed by typical whole blood filtration of selected membranes. The data presented highlight that BSA moieties do not particularly affect the white blood cell (WBC) adhesion and platelet adhesion of EVAL membranes whereas resulted in a significantly higher red blood cell (RBC) recovery and decreased hemolysis under in vitro conditions. Accordingly, BSA-immobilized EVAL membranes were made to asymmetric leukodepletion filters; however, their further evaluation by whole blood filtration ensued that immobilization of BSA could not particularly enhance the overall whole blood leukodepletion efficacy of EVAL membranes.

CHANGES IN THE MORPHOLOGIC STATE OF RED BLOOD CELLS IN PATIENTS WITH POLYTRAUMA

The measurement of RBC indices is of particular importance as an indirect intermediate component of the recovery process after exposure to hypoxia, ischemia, hypothermia in patients with traumatic disease due to polytrauma. The aim of this study was to evaluate the parameters of the morphologic state of red blood cells as a reaction of patients with traumatic disease. Materials and methods.A prospective study was carried out evaluation of 120 patients suffering from polytrauma. The patients were divided into 3 groups according to the principle of using additional substances as a part of IC. The parameters of morphologic state of red blood cells were studied. Results and discussion. The course of acute and early periods of traumatic disease has accompanied variability morphological forms of red blood cells, as evidenced by a decrease in MCV and RDW increased levels of the 3rd to 5th day. The optimized therapy used in the treatment of patients, has a positive effect on the parameters of red blood cells, contributes to a better recovery of red blood cells after hypoxia resulting multiple trauma. Conclusion. The study has demonstrated variations in the morphologic properties of red blood cells in the acute and early period of traumatic disease, as confirmed by pathological changes of the erythrocyte indices. The administration of the proposed therapy with the use of D-fructose-1.6-diphosphate sodium salt of hydrate has a positive effect on the parameters of red blood cells, contributes to a better recovery after hypoxia resulting multiple trauma. Given this, the calculations of the red blood cell indices may carry additional diagnostic information, which allows identify trends for not obvious pathological changes and quality of treatment.