Transfusion Recovery Research Articles

Frequencies of blood donation and types of blood donors on storage lesions as assessed by antioxidants and lipid peroxidation levels

Introduction: Influence of Blood storage lesions on Red Blood Cells transfusion recovery and therapeutic efficacies have been well documented. Several stabilizing additives to counteract or minimize these untoward changes have been formulated. Despite these stabilizers, detectable hemolysis occurs in the blood stored in the blood bank hence, need for search on other contributing factors. Assessment of the effects of Blood Donation Frequencies and Types of Blood Donors on Storage Lesions as Assessed by the Level of Antioxidants and Lipid Peroxidation. Materials and Methods: Ethically approved cross sectional experimental research on a total numbers of 120 recruited male of family replacement donors (n=30) and Remunerated donors (n=90). Remunerated donors were grouped into three (3) based on the frequency of donation per year as mild (n=30), moderate (n=30) and high frequency (n=30) donors. Four hundred and fifty milliliters (450 ml) of blood drawn from each donor into double blood bag containing CPDA-1 anticoagulant. 100 ml of well mixed blood was transferred into the satellite bag, detached and stored in the blood bank at 2-6 C for 35 days. The blood samples analyzed for MDA, TAP, URIC, and GSH at 0, 7, 14, 21, 28 and 35 days of storage. Results: In the recruited donors, majority were above 40 years of age where family donors had secondary education, government employed; feed majorly on carbohydrate, none alcohol drinkers, none cigarette smokers with history of donating more than four years before the study and less than four times donation frequency per year. Remunerated blood donors had primary education, self-employed, moderate alcohol drinkers and moderate cigarette smokers donating more than four times a year and more than four years donation experience. In this study, the GPX, TAP and Uric acids for family were higher than remunerated at baseline and the values decrease across the week for both categories. MDA value for family was lower than remunerated donors at baseline and the values increase across the week for both categories. Conclusion: This study established variation in oxidant and antioxidants levels in whole blood stored up to 5 weeks influenced by Blood Donation Frequencies and Types of Blood Donors on Storage Lesions.

Storage-Induced Micro-Erythrocytes Can Be Quantified and Sorted by Flow Cytometry.

Refrigerated storage of red cell concentrates before transfusion is associated with progressive alterations of red blood cells (RBC). Small RBC (type III echinocytes, sphero-echinocytes, and spherocytes) defined as storage-induced micro-erythrocytes (SME) appear during pretransfusion storage. SME accumulate with variable intensity from donor to donor, are cleared rapidly after transfusion, and their proportion correlates with transfusion recovery. They can be rapidly and objectively quantified using imaging flow cytometry (IFC). Quantifying SME using flow cytometry would further facilitate a physiologically relevant quality control of red cell concentrates. RBC stored in blood bank conditions were stained with a carboxyfluorescein succinimidyl ester (CFSE) dye and incubated at 37°C. CFSE intensity was assessed by flow cytometry and RBC morphology evaluated by IFC. We observed the accumulation of a CFSEhigh RBC subpopulation by flow cytometry that accounted for 3.3 and 47.2% at day 3 and 42 of storage, respectively. IFC brightfield images showed that this CFSEhigh subpopulation mostly contains SME while the CFSElow subpopulation mostly contains type I and II echinocytes and discocytes. Similar numbers of SME were quantified by IFC (based on projected surface area) and by flow cytometry (based on CFSE intensity). IFC and scanning electron microscopy showed that ≥95% pure subpopulations of CFSEhigh and CFSElow RBC were obtained by flow cytometry-based sorting. SME can now be quantified using a common fluorescent dye and a standard flow cytometer. The staining protocol enables specific sorting of SME, a useful tool to further characterize this RBC subpopulation targeted for premature clearance after transfusion.

Rapid clearance of storage-induced microerythrocytes alters transfusion recovery

AbstractPermanent availability of red blood cells (RBCs) for transfusion depends on refrigerated storage, during which morphologically altered RBCs accumulate. Among these, a subpopulation of small RBCs, comprising type III echinocytes, spheroechinocytes, and spherocytes and defined as storage-induced microerythrocytes (SMEs), could be rapidly cleared from circulation posttransfusion. We quantified the proportion of SMEs in RBC concentrates from healthy human volunteers and assessed correlation with transfusion recovery, investigated the fate of SMEs upon perfusion through human spleen ex vivo, and explored where and how SMEs are cleared in a mouse model of blood storage and transfusion. In healthy human volunteers, high proportion of SMEs in long-stored RBC concentrates correlated with poor transfusion recovery. When perfused through human spleen, 15% and 61% of long-stored RBCs and SMEs were cleared in 70 minutes, respectively. High initial proportion of SMEs also correlated with high retention of RBCs by perfused human spleen. In the mouse model, SMEs accumulated during storage. Transfusion of long-stored RBCs resulted in reduced posttransfusion recovery, mostly due to SME clearance. After transfusion in mice, long-stored RBCs accumulated predominantly in spleen and were ingested mainly by splenic and hepatic macrophages. In macrophage-depleted mice, splenic accumulation and SME clearance were delayed, and transfusion recovery was improved. In healthy hosts, SMEs were cleared predominantly by macrophages in spleen and liver. When this well-demarcated subpopulation of altered RBCs was abundant in RBC concentrates, transfusion recovery was diminished. SME quantification has the potential to improve blood product quality assessment. This trial was registered at www.clinicaltrials.gov as #NCT02889133.

The Effect of Washing of Stored Red Blood Cell Transfusion Units on Post Transfusion Recovery and Outcome in a Pneumosepsis Animal Model.

Septic patients are often anemic, requiring red blood cell (RBC) transfusions. However, RBC transfusions are associated with organ injury. The mechanisms of RBC-induced organ injury are unknown, but increased clearance of donor RBCs from the circulation with trapping in the organs could play a role. We hypothesized that washing of RBCs prior to transfusion may reduce clearance and trapping of donor cells and thereby reduce organ injury. Sprague-Dawley rats were inoculated intratracheally with 10 colony-forming units (CFU) of Streptococcus pneumoniae or vehicle as a control and transfused with either a washed or standard (non-washed) biotinylated RBC transfusion from syngeneic rats. Controls received saline. Blood samples were taken directly after transfusion and at 24 h to calculate the 24 h post transfusion recovery (PTR). After sacrifice, flow cytometry was used to detect donor RBCs in organs and blood. The organs were histologically scored by a pathologist and CFUs in the lung and blood were counted. The 24h-PTR was similar between healthy and pneumoseptic rats after a standard transfusion. In healthy rats, a washed transfusion resulted in a higher PTR and less accumulation of donor RBCs in the organs compared with a standard transfusion. However, during pneumonia, this effect of washing was not seen. Transfusion did not further augment lung injury induced by pneumonia, but washing decreased bacterial outgrowth in the lungs associated with reduced lung injury. In healthy recipients, washing increased 24h-PTR of donor RBCs and decreased trapping in organs. In pneumoseptic rats, washing reduced bacterial outgrowth and lung injury, but did not improve PTR.

Post-transfusion platelet responses in critically ill cancer patients with hypoproliferative thrombocytopenia.

Platelet transfusion is aimed at increasing platelet counts to prevent or treat bleeding. Critically ill cancer patients with hypoproliferative thrombocytopenia are high consumers of blood products. We herein described their post-transfusion platelet responses in the intensive care unit (ICU) and analyzed the determinants of poor post-transfusion increments. This was a single-center 9-year (2009-2017) retrospective observational study. Patients with malignancies and presumed or proven hypoproliferative thrombocytopenia who had received at least one platelet transfusion in the ICU were included. Poor post-transfusion platelet increments were defined as body surface-adjusted corrected count increment (CCI) <7, or alternatively as weight-adjusted platelet transfusion recovery (PTR) <0.2. Patients were deemed refractory to platelet transfusions when two consecutive ABO-compatible transfusions resulted in poor platelet increments. A total of 1470 platelet transfusions received by 326 patients were analyzed. Indications for platelet transfusions were distributed into prophylactic (44.5%), peri-procedural (18.1%) and therapeutic (37.4%). Regardless of indications, 54.6% and 55.4% of transfusion episodes were associated with a CCI <7 or a PTR <0.2. Factors independently associated with poor post-transfusion increments were lower body mass index, spleen enlargement, concurrent severity of clinical condition, fever ≥39°C, antibiotic therapy and increased storage duration of platelet concentrates. Eventually, 48 patients developed refractoriness to platelet transfusion, which was associated increased incidence of bleeding events. Platelet transfusions are often associated with poor increments in critically ill cancer patients with hypoproliferative thrombocytopenia. The findings suggest amenable interventions to improve the platelet transfusion practices in this setting.

Storage-Induced Micro-Erythrocytes Are Rapidly Cleared from Recipient Circulation and Predict Transfusion Recovery

Background Hypothermic storage of red blood cell (RBC) concentrates for up to 42 days is associated with biochemical, molecular, morphological, and mechanical modifications. This "storage lesion" increases with storage duration and is associated with increased clearance of transfused storage-damaged RBCs from the recipient's circulation in the first few hours post-transfusion. This rapid clearance reduces transfusion efficacy, but how it occurs is not fully elucidated. RBCs with reduced surface area called "storage-induced micro-erythrocytes" (SMEs) were recently described. Their proportion increases from 2% to 23% during storage. Their reduced surface-to-volume ratio is expected to induce rapid mechanical clearance by the spleen. We aimed to evaluate whether SMEs can be used as a marker of transfusion efficacy, if this subpopulation of RBCs is preferentially cleared by the spleen after transfusion, and if so, by which mechanisms. Methods We evaluated the proportion of SMEs in stored RBC concentrates in vitro using ImageStream and correlated it to the 51Chromium-labeled 24h post-transfusion recovery (24hPTR) in vivo in 31 healthy human volunteers. We then investigated the fate of SMEs during 8 ex vivo perfusions of human spleens (16 RBC concentrates stored for 35-42 days). Finally, we developed a mouse transfusion model to assess the fate of SMEs in vivo and determine their main mechanisms of clearance. Results The proportion of SMEs in RBC concentrates at day 42 of storage correlated negatively with 24hPTR in healthy volunteers (r=-0.42, P&amp;lt;0.01). When perfused ex vivo into human spleens, 15% of stored RBCs (35-42 days of storage) were cleared during the first 40 min of perfusion in a 2-step process: 7% of circulating RBCs disappeared in the first 2 min (1-2 passages through the spleen) while 8% were cleared between 10 and 40 min after initiating perfusion (&amp;gt;5 passages through the spleen). The percentage of SMEs correlated with splenic retention rate ex vivo (r=0.46, p&amp;lt;0.05). Morphological analysis of 6 stored RBC concentrates showed a mean decrease in the proportion of SMEs from 20.2% to 7.8% between the beginning and end of splenic perfusions. In our mouse transfusion model, SMEs accumulated during RBC storage. The 24hPTR also decreased with storage duration (64% on Day 14 vs. 95% on Day 1). The decrease in 24hPTR of long-stored RBCs was mostly due to clearance of the SME subpopulation. SME and morphologically normal long-stored RBC subpopulations displayed clearances of 83% and 13%, respectively. Stored RBCs accumulated predominantly in the spleen post-transfusion, and were mainly ingested by macrophages. In macrophage-depleted mice, 24hPTR improved (from 64% to 79%), splenic accumulation and clearance of SMEs were delayed, and the proportion of inflammatory monocytes increased and mediated clearance. In splenectomized mice, clearance of SMEs was not delayed, but increased accumulation was observed in the liver and bone marrow, and increased erythrophagocytosis by inflammatory monocytes was also observed. Conclusions We show that the proportion of SMEs correlates with 24hPTR in healthy human volunteers and with retention in human spleens perfused ex vivo. In vivo mouse data confirms these findings, showing that SMEs are cleared from the recipient circulation during the 24h following transfusion. Clearance of SMEs is delayed in macrophage-depleted mice, suggesting a central role of macrophages in this process. The human spleen is also likely to clear SMEs from the recipient's circulation, as suggested by experiments with human spleens perfused ex vivo. However, the spleen is not required, because SME clearance is not affected in splenectomized mice. This suggests that other organs may compensate to remove SMEs and highlights the importance of eliminating these morphologically-altered RBCs. Finally, quantification of SMEs is an operator-independent, reproducible marker of transfusion efficacy. It can be used to assess the potential of new processes to prepare and store RBC concentrates. Pre-transfusion quantification of SMEs could benefit chronically transfused patients, for whom improved transfusion efficacy is expected to reduce transfusion-induced iron overload. Disclosures Roussel: Zimmer Biomet: Research Funding. MARIN:Zimmer Biomet: Research Funding. Spitalnik:Hemanext: Membership on an entity's Board of Directors or advisory committees; Tioma, Inc.: Consultancy. Hermine:AB science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Research Funding. Buffet:Zimmer Biomet: Research Funding. Amireault:Zimmer Biomet: Research Funding.

Measuring Post-transfusion Recovery and Survival of Red Blood Cells: Strengths and Weaknesses of Chromium-51 Labeling and Alternative Methods.

The proportion of transfused red blood cells (RBCs) that remain in circulation is an important surrogate marker of transfusion efficacy and contributes to predict the potential benefit of a transfusion process. Over the last 50 years, most of the transfusion recovery data were generated by chromium-51 (51Cr)-labeling studies and were predominantly performed to validate new storage systems and new processes to prepare RBC concentrates. As a consequence, our understanding of transfusion efficacy is strongly dependent on the strengths and weaknesses of 51Cr labeling in particular. Other methods such as antigen mismatch or biotin-based labeling can bring relevant information, for example, on the long-term survival of transfused RBC. These radioactivity-free methods can be used in patients including from vulnerable groups. We provide an overview of the methods used to measure transfusion recovery in humans, compare their strengths and weaknesses, and discuss their potential limitations. Also, based on our understanding of the spleen-specific filtration of damaged RBC and historical transfusion recovery data, we propose that RBC deformability and morphology are storage lesion markers that could become useful predictors of transfusion recovery. Transfusion recovery can and should be accurately explored by more than one method. Technical optimization and clarification of concepts is still needed in this important field of transfusion and physiology.

Resuscitation with Anaerobically Stored Blood

BackgroundHemorrhagic shock (HS) severely decreases oxygen (O2) delivery and induces cardiovascular collapse. In parallel to controlling the hemorrhage, doctors respond by infusing large volumes of red blood cells (RBCs) to restore blood volume, O2 carrying capacity, and hemodynamic stability. The quality of the transfused RBCs determines the recovery from HS, and extent of sequelae prompted by the HS. This study compares the ability to recover from HS with conventionally stored RBCs, anaerobically (O2 saturation &lt;10%) stored RBCs, and anaerobic/hypercapnic (O2 saturation &lt;10% &amp; pCO2 (@37°C) ~70 mmHg) stored RBCs.MethodsPacked red blood cells (pRBCs) stored in AS‐3 after leukorfiltration were created from donor rats. pRBC were randomly stored under 1) conventional; 2) anaerobic; 3) anaerobic/hypercapnic conditions. Sprague‐Dawley rats (150–200g) were hemorrhaged (50% of blood volume), held in hypovolemia for 30 minutes, and resuscitated to recover blood pressure to 90% pre‐hemorrhage with pRBC stored for 3 weeks. Systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation. Vital organ inflammation, oxygenation, and function were evaluated post resuscitation. Data were analyzed using two‐way ANOVA, followed by the appropriate post hoc analyses.ResultsConventionally stored RBCs were susceptible to both biochemical and mechanical changes during storage compared to anaerobic and anaerobic/hypercapnic storage. 24‐hour post transfusion recovery of anaerobically and anaerobic/hypercapnic stored RBCs was significantly higher compared to conventionally stored RBCs (P&lt;0.05). Shock impaired cardiac function and oxygen delivery and resuscitation with pRBCs restored cardiac output. Resuscitation with conventionally stored cells resulted in severe cardiac ischemia. Resuscitation with either anaerobic or anaerobic/hypercapnic required significantly lower volume (1.9 and 2 fewer units than conventional storage) of pRBC to preserve hemodynamics during resuscitation (P &lt; 0.05). Animals resuscitated with conventionally stored RBCs showed impaired ability to deliver O2 to tissues, increased lung, liver, and spleen inflammatory markers, and decreased liver and kidney function, compared to animals resuscitated with either anaerobic or anaerobic/hypercapnic pRBC.ConclusionStudies indicate that anaerobic or anaerobic/hypercapnic storage of RBC can improve clinical outcomes from hemorrhagic shock in a rat model, as evidenced by hemodynamic stability, O2 delivery, and vital organ inflammation and function.Support or Funding InformationNIH Grants from the National Heart Lung and Blood Institute, T32‐HL105373, P01‐HL110900, R01‐HL126945. Financial support was received for Hemanext for the completion of the study. Hemanext did not participate in the implementation of the study.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transfusion sanguine chez les personnes âgées : étude descriptive à propos de 241 patients au CHRU de Brest

Red blood cell transfusion is commonly required for elderly patients with little specific guidelines for this population. We wished to study the transfusion practices for patients aged≥75 years transfused with red blood cells in all units of our institution. The aim of this work is to describe this population of patients and to compare the transfusion practices to the HAS and ANSM guidelines published in 2014. MethodsAn observational study was performed including patients≥75 years, transfused with red blood cells between 27th January to 27th March 2014. Analysis of data included the patients’ typology, the characteristics of anemia, the indications and transfusion practices, and the adequation with the guidelines. ResultsTwo hundred and forty-one patients were transfused during the period with 422 transfusion episodes (mean age 83±5.2 years, sex-ratio women/men 1.4 with 4.4±2 pathologies and 6.7±3.2 treatments). Only 4.5% of the patients were transfused in geriatric wards. In 61% of the cases, the anemia is acute and chronic in 39%. Fatigue was reported in 50.6% of the cases and dyspnee in 31.7%. A percentage of 31.1 of the patients were prescribed furosemide for transfusion. The mean transfusion recovery was 1.2±0.5g/dL. Two adverse events were reported. Overall, only 10.4% of the prescriptions were not compliant with the guidelines of 2014. ConclusionIn this study, we describe transfusion practices for patients aged≥75 years. Although the non-compliance with the guidelines is weak, it is possible to improve the transfusion practices taking care of the geriatric characteristics. Prospective studies on a larger scale have to be done to converge on a more consensual management.

Efficiency of ring butterfly sewing in lower uterine segment aided with tourniquet in treating pernicious placenta previa combined with placenta percreta

Objective To investigate the efficiency of ring butterfly sewing in lower uterine segment aided with tourniquet in treating pernicious placenta previa combined with placenta percreta. Methods Twelve pregnant women diagnosed with pernicious placenta previa combined with placenta percreta by prenatal ultrasound in Peking University First Hospital from April 1, 2012 to November 30, 2014, were enrolled. All of them received elective cesarean delivery using ring butterfly sewing in lower uterine segment aided with tourniquet. The efficiency of this novel technique, in term of blood loss and vital signs during operation, blood transfusion, and postoperative recovery, was analyzed. Results Twelve cases were diagnosed with placenta percreta during operation, with placenta invading the perimetrium in seven cases and urinary bladder being involved in one case. Ring butterfly sewing in lower uterine segment aided with tourniquet during elective cesarean delivery was all successful in the 12 cases. The median amount of blood loss was 1 000(400-2 000) ml during operation and the amount of blood loss was lower than 1 000 ml in three cases. Two cases received no blood transfusion, and the median blood transfusion in the other 10 cases were 400(400-1 200) ml. The mean operation time was (75±22) min (43-131) min. Eleven cases had normal lochia and temperature after operation and the other one had placenta invading urinary bladder and partial placenta in situ, and developed fever at day 3 after operation, but recovered after antibiotic treatment, and no placenta tissue was seen by ultrasound at day 59 after operation. Conclusions The ring butterfly sewing in lower uterine segment aided with tourniquet is highly effective for the pernicious placenta previa combined with placenta percreta, and it is a simple, fast and fertility sparing technique with low blood loss and without usage of special equipment. Key words: Placenta previa; Placenta percreta; Tourniquets; Ligation; Suture techniques; Gynecologic surgical procedures

Transfusion of fresh vs. older red blood cells in the context of infection.

The red blood cell (RBC) storage interval has been extended from less than a week to the current storage interval of 6-8 weeks. Regulatory criteria for extending storage rely upon a minimal degree of hemolysis and acceptable in vivo 24-h post transfusion recovery. Clinical studies of safety and efficacy have never been required. Concerns have arisen that RBC toward the end of storage develop a 'storage lesion' with previously unrecognized toxicity. Of the several mechanisms proposed, the bolus of iron delivered to macrophages as a result of hemolysis of stored RBC might pose a particular risk to patients with existing infections. We developed a canine model of pneumonia to compare the toxicity of stored RBC transfusion. We described increased mortality after transfusion of old RBC. We found that transfused older RBC increased mortality, in vivo hemolysis, circulating cell-free hemoglobin that scavenges nitric oxide, and elevations of non-transferrin bound and plasma labile iron. Disappearance of circulating iron correlated with increased mortality, worsening pulmonary function, and bacterial proliferation. Washing decreased the mortality associated with transfusing older RBC, but had the opposite effect on fresher blood. With low doses of bacteria, survival was unaffected by the age of blood, whereas high bacteria doses masked any effect of RBC age on mortality. Older RBC may have adverse effects, but the patient's clinical status, the age, volume and method of preparation of the RBC may be critical variables. Several mechanisms may account for this toxicity, but in the presence of bacterial infection, availability of iron likely plays a major role.

Neonatal Polycythemia Secondary to Twin to Twin Transfusion Syndrome- A Case Report

Twin-twin transfusion syndrome (TTTS) is one of the most serious complications of monochorionic multiple gestations in which the receipient twin becomes plethoric while the donor twin becomes anaemic. We report the case of the 1st of a set of male twins who presented on the 2nd day of life with excessive ruddiness of the whole body noted at birth and yellowness of the eyes and face noted on the 2nd day of life. Given the comparative clinicolaboratory findings, a diagnosis of neonatal polycythemia secondary to twin to twin transfusion syndrome was made. Post partial exchange blood transfusion (PET) recovery was remarkable. This report was made because of its rarity and to raise the index of suspicion for such cases to enable proper evaluation and management.

Profil clinico-biologique et immunohématologique des patients atteints de β-thalassémie en Tunisie : à propos de 26 cas

Aim of the studyTo study the clinical and biological profile of β-thalassemic patients in our region, reflecting the quality of their care. Patients and methodsA retrospective study (2010–2011) on 26 β-thalassemic patients followed in the pediatrics service at CHU Farhat Hached Sousse, Tunisia. Epidemiological, clinical and biological data were collected from medical records and transfusion files of patients. The transfusion protocol adopted was to maintain a hemoglobin level>10g/dL by regular transfusions every 3–4 weeks. Iron chelation therapy, in order to maintain serum ferritin<1500ng/mL, was introduced when serum ferritin exceeded 800–1000ng/mL. ResultsThe mean age of patients at diagnosis was 15 months. The clinical impact of anemia had resulted in failure to thrive in 54% of patients and facial dysmorphism in 23%. The average transfusion requirement was estimated at 311.02mL/kg/year with 6 cases of hyperconsumption. The immunohaematological monitoring showed the appearance of anti-RBC alloimmunization in one patient and 4 cases of autoimmunization. Poor adherence of chelation therapy was 62% and causing 5 cases of cardiac complications, 4 cases of liver injury and 14 cases of endocrine complications. ConclusionImproving the therapeutic care of β-thalassemic children requires better monitoring of transfusion recovery and improved adherence to chelation therapy.

Metalloproteinase Regulation Determines Integrin αIIbβ3 Function and Enhances the Capacity of ES Cell-Derived Platelets.

To resolve the lack of sources for products used in blood transfusion therapy, the utilization of embryonic stem cells (ESCs) has been proposed. However, the use of ESC-derived cells has potential drawbacks including, immunological rejection and teratoma formation. Being fully differentiated cells lacking a nucleus, platelets can be irradiated to eliminate passenger ESCs and other lineages before transfusion. We previously established in vitro differentiation system whereby proplatelet-producing megakaryocytes can be derived from murine ESCs on OP9 stroma, but the platelets were generated with a low efficiency and exhibited weak agonist-induced integrin αIIbβ3 activation and spreading on immobilized fibrinogen. Here, we describe an improved method to obtain ESC-derived platelets of potential use for hemostasis and thrombosis in vivo. We generated a novel murine ESC line wherein the GPIbα promoter regulates EGFP expression as a marker (GP-G ESCs). The combination of flow cytometry with GP-G-derived embryoid bodies and OP9 co-culture enabled us to enrich integrin αIIb + c-kit + CD9 + Sca-1 − progenitors on day 5–6 of culture as a major fraction capable of differentiating into GFP+ mature megakaryocytes on day 8–10 in the presence of TPO. This strategy increased platelet production by 10-fold (P<0.01) compared to our previous report (PNAS, 2002, 99:12819). Interestingly, αIIb+platelets included 35–40% GPIbα-negative population besides GPIbα-positive on day 13–15, which was caused by an increased activity of matrix metalloproteinase (MMP) and TACE (ADAM17) to shed GPIbα in culture. Inhibition of MMP and TACE with GM6001 or TAP1 delayed the clearance rate on post transfusion recovery in vivo as transfused into a platelet-depleted mouse model. Maintaining GPIbα expression in the presence of MMP/TACE inhibitors restored the specific fibrinogen-bound to integrin αIIbβ3 and platelet spreading on fibrinogen as evidenced by the generation of filopodia and lamellipodia. Collectively, these results suggest that 1) ESC-derived αIIb+ c-kit + CD9+ Sca-1 − progenitors may contribute to enrich megakaryocytopoiesis at high efficiency; 2) inhibition of metalloproteinase activity to retain GPIbα expression is required for αIIbβ 3 integrin inside-out and outside-in signaling in ESC-derived platelets; and 3) these platelets may be useful alternatives for future clinical application.

Determinants of ABH expression on human blood platelets

AbstractPlatelets express ABH antigens, which can adversely effect platelet transfusion recovery and survival in ABH-incompatible recipients. To date, there has been no large, comprehensive study comparing specific donor factors with ABH expression on platelet membranes and glycoconjugates. We studied ABH expression in 166 group A apheresis platelet donors by flow cytometry, Western blotting, and thin layer chromatography relative to donor age, sex, A1/A2 subgroup, and Lewis phenotype. Overall, A antigen on platelet membranes, glycoproteins, and glycosphingolipids was linked to an A1 red blood cell (RBC) phenotype. Among A1 donors, platelet ABH varied significantly between donors (0%-87%). Intradonor variability, however, was minimal, suggesting that platelet ABH expression is a stable, donor-specific characteristic, with 5% of A1 donors typing as either ABH high- or low-expressers. Group A2 donors, in contrast, possessed a Bombay-like phenotype, lacking both A and H antigens. Unlike RBCs, ABH expression on platelets may be determined primarily by H-glycosyltransferase (FUT1) activity. Identification of A2 and A1 low expressers may increase the availability and selection of crossmatched and HLA-matched platelets. Platelets from group A2 may also be a superior product for patients undergoing A/O major mismatch allogeneic progenitor cell transplantation. (Blood. 2005;105:3356-3364)

Modulation of Idiotypic and Antiidiotypic Immunoglobulin G Responses in an Alloimmune Thrombocytopenic Patient Associated with Extracorporeal Protein A Immunoadsorption

In the present case study, a patient with Non-Hodgkin. Lymphoma underwent combination chemotherapy resulting in severe pancytopenia requiring transfusion support with blood products. The patient became refractory to random donor platelet transfusions and subsequently received five immunoadsorption treatments. The patient's clinical response to immunoadsorption therapy was assessed by monitoring platelet transfusion recovery and survival. In addition, changes in antibody responses were assessed. Early during the course of immunoadsorption therapy, antiplatelet immunoglobulin G (IgG) alloantibody was detected. There was a decline in antiplatelet IgG alloantibody levels by the last immunoadsorption treatment associated with increases to platelet correct count increments after completion of immunoadsorption therapy. In addition, elevated levels of antiidiotypic IgG antibody detected early during the course of therapy were significantly reduced by the last immunoadsorption treatment. This case study suggests that specific alloimmune idiotypic IgG antibody and corresponding antiidiotypic IgG antibody responses may be modulated in association with extracorporeal immunoadsorption employing protein A/silica columns.

The Expression of IgG Allotypes on Platelets and Immunization to IgG Allotypes in Multitransfused Thrombocytopenic Patients

We investigated whether the platelet-membrane surface carries IgG allotypic antigens and whether these determinants may be important in platelet transfusion therapy. Using a hemagglutination inhibition assay, we showed that the G1m IgG allotypes (a, x, f) and K1m and K3m light-chain allotypes are expressed on the surface of platelets, whereas G3m allotype antigenic determinants were not detectable. In 146 multitransfused thrombocytopenic patients, 35 (24%) patients were found to have antiallotypic antibodies. To study the effect of antiallotypic antibodies on platelet transfusion outcome, patients received platelet transfusions from donors, either positive or negative for the IgG allotype to which patients were immunized. Of the 19 antigen-positive and 19 antigen-negative platelet transfusions given, respectively, the mean platelet count increments at 1 hour were 8,402 +/- 6,402 +/- 6,721 (1 SD) and 9,799 +/- 5,559 (1 SD) P less than .2. Transfusion reactions were not more common when antigen-positive platelet transfusions were given. Despite the presence of IgG allotypic determinants on platelets, allotypic antibodies do not decrease platelet transfusion recovery. Furthermore, passive administration of plasma containing IgG allotypes to patients with antiallotypic antibodies does not lead to innocent bystander-mediated platelet destruction.

The expression of IgG allotypes on platelets and immunization to IgG allotypes in multitransfused thrombocytopenic patients

We investigated whether the platelet-membrane surface carries IgG allotypic antigens and whether these determinants may be important in platelet transfusion therapy. Using a hemagglutination inhibition assay, we showed that the G1m IgG allotypes (a, x, f) and K1m and K3m light-chain allotypes are expressed on the surface of platelets, whereas G3m allotype antigenic determinants were not detectable. In 146 multitransfused thrombocytopenic patients, 35 (24%) patients were found to have antiallotypic antibodies. To study the effect of antiallotypic antibodies on platelet transfusion outcome, patients received platelet transfusions from donors, either positive or negative for the IgG allotype to which patients were immunized. Of the 19 antigen-positive and 19 antigen-negative platelet transfusions given, respectively, the mean platelet count increments at 1 hour were 8,402 +/- 6,402 +/- 6,721 (1 SD) and 9,799 +/- 5,559 (1 SD) P less than .2. Transfusion reactions were not more common when antigen-positive platelet transfusions were given. Despite the presence of IgG allotypic determinants on platelets, allotypic antibodies do not decrease platelet transfusion recovery. Furthermore, passive administration of plasma containing IgG allotypes to patients with antiallotypic antibodies does not lead to innocent bystander-mediated platelet destruction.

Etude in vitro de la conservation de plaquettes pendant 5 jours dans des poches d'épaisseur réduite

An in vitro study of platelet concentrates storage for 5 days was performed in PVC bags. Modification of the original three-day containers were introduced by thickness reduction. The results at fifth day were comparable to those at third day in standard plastic bags. During storage, variations of platelet counts were very slight with a low LDH release. PH was stable with a good maintenance of phase microscope platelet morphology. PCO2 and PO2 measurements showed a satisfactory gas permeability which could explain a limited lactate production. If in vivo studies of transfusion recovery confirm these data, platelet concentrates storage could be extended up to 5 days by such modifications of standard three-day PVC bags.

In vitro function and post-transfusion survival of granulocytes collected by continuous-flow centrifugation and by filtration leukapheresis

The function of granulocytes collected by continuous-flow centrifugation (CFC) and by filtration leukapheresis (FL) was studied in vitro, and the post-transfusion recovery and intravascular survival of these cells was studied by autologous transfusion in normal donors. Granulocytes collected by both FL and CFC leukapheresis (CFCL) functioned normally in the quantitative nitroblue tetrazolium, oxygen consumption, and chemotaxis assays. Bacterial killing was slightly but consistently decreased in FL but not CFCL granulocytes. The post- transfusion recovery of control granulocytes collected by ordinary phlebotomy averaged 52% in eight transfusions, compared with 34% for six CFCL granulocyte concentrates and 16% for six FL concentrates. The intravascular half-times were 3.8 hr for phlebotomy and 3.0 hr for CFCL granulocytes. FL granulocytes had survival curves which were nonlinear and a single half-life could not be calculated. The average half-time 30 min after transfusion was 1.3 hr, and 3 hr after transfusion it was 2.6 hr. Granulocytes collected by FL had a mild impairment of bacterial killing, decreased post-transfusion recovery, and altered intravascular kinetics. None of these abnormalities was found in granulocytes collected by CFCL.