Washed Blood Cells Research Articles

Perbedaan Hasil Derajat Aglutinasi Serum Grouping Tube Test Dengan Suspensi Reagen NaCl 0,9% Siap Pakai dan Suspensi Reagen NaCl 0,9% Dari Garam Dapur

ABO blood group examination is a simple laboratory test performed to determine a person’s blood group. There are two types of ABO blood group test, namely cell grouping and serum grouping. Serum grouping examination is very important to prevent incompatible transfusion reactions and organ transplant rejection. Serum grouping was carried out using 10% saline wash suspension of cell A, B and O. The blood cell washing stage was used to remove fragile cells and interfering substances that could result in errors in the examination. The main ingredient used in the blood cell washing procedur is 0.9% NaCl. This study aims to determine the degree of agglutination serum grouping tube test with ready to use 0.9% NaCl reagent suspension and 0.9% NaCl reagent suspension from table salt. The type of research is an experiment. The study was conducted at UDD PMI South Sumatra Province on 30 December 2021 to 5 January 2022 with a totalsample of 67 samples. The results showed that the average degree of agglutination the serum grouping tube test with 0.9% NaCl reagent ready to use was 3.73 and the average degree of agglutination the serum grouping tube test with0.9% NaCl reagent from table salt was 3.69. Wilcoxon test results obtained p value = 0.450. The conclusion of the study, that there is no differences in the results of the agglutination serum grouping tube test with ready to use 0.9% NaCl reagen suspension and 0.9% NaCl reagent suspension from table salt. The table salt showed that 0.9% NaCl reagent can be used as alternative reagent NaCl 0.9% ready to use in the examination of Iserum grouping tube test.

Isolation of Culicoides- and Mosquito-Borne Orbiviruses in the Southwestern Islands of Japan Between 2014 and 2019.

The circulation of arboviruses in livestock ruminants has often gone unrecognized owing to the fact that a significant percentage of arboviruses probably induce subclinical infections and/or negligible symptoms in infected animals. To determine the current situation of arbovirus circulation in the Yaeyama Islands, attempts to isolate viruses from bovine blood samples collected between 2014 and 2019 have been made. In total, 308 blood samples were collected during the study period, and 43 of them induced cytopathic effects (CPEs) in cell cultures. The identification of the CPE agents was performed by reported RT-PCR assays and a high-throughput analysis with a next-generation sequencing platform. The obtained viruses consisted of an orthobunyavirus (Peaton virus), Culicoides-borne orbiviruses (bluetongue virus serotypes 12 and 16, epizootic hemorrhagic disease virus [EHDV] serotypes 5, 6, and 7, D'Aguilar virus, and Bunyip Creek virus), and potential mosquito-borne orbiviruses (Yunnan orbivirus, Guangxi orbivirus, and Yonaguni orbivirus). Most of the orbiviruses were recovered from washed blood cells with mosquito cell cultures, suggesting that this combination was more efficient than other combinations such as plasma/blood cells and hamster cell lines. This marked the first time that the isolation of EHDV serotypes 5 and 6 and three potential mosquito-borne orbiviruses was recorded in Japan, showing a greater variety of orbiviruses on the islands than previously known. Genetic analysis of the isolated orbiviruses suggested that the Yaeyama Islands and its neighboring regions were epidemiologically related. Some of the viruses, especially the potential mosquito-borne orbiviruses, were isolated during several consecutive years, indicating their establishment on the islands.

Isolation of epizootic hemorrhagic disease virus serotype 7 from cattle showing fever in Japan in 2016 and improvement of a reverse transcription-polymerase chain reaction assay to detect epizootic hemorrhagic disease virus.

Epizootic hemorrhagic disease (EHD) is an arthropod-borne disease of wild and domestic ruminants caused by the EHD virus (EHDV). To date, seven EHDV serotypes have been identified. In Japan, strain Ibaraki of EHDV serotype 2 has caused outbreaks of Ibaraki disease in cattle. In addition, EHDV serotype 7 (EHDV-7) has caused large-scale EHD epizootics. In mid-September 2016, eight cattle at a breeding farm in Fukuoka Prefecture, Japan developed fever. Since EHDV-7 was detected in sentinel cattle in western Japan in 2016, we suspected that the cause of this fever might be an EHDV-7 infection. In this study, we tested cattle for EHDV-7 and some other viruses. Consequently, EHDV was isolated from washed blood cells collected from three of the eight cattle, and genetic analysis of genome segment 2 revealed that this isolate was EHDV-7. Moreover, all affected cattle tested positive for anti-EHDV-7 neutralizing antibodies. Our results suggest that the fever was caused by EHDV-7 infection. In addition, we modified a conventional reverse transcription polymerase chain reaction assay for the specific detection of EHDV. This modified assay could detect various strains of EHDV isolated in Japan, Australia, and North America. Furthermore, the assay permitted the detection of EHDV-7 in blood cells collected from seven of the eight cattle. We believe that this modified assay will be a useful tool for the diagnosis of EHD.

Minimizing isotropic and deviatoric membrane energy - An unifying formation mechanism of different cellular membrane nanovesicle types.

Tiny membrane-enclosed cellular fragments that can mediate interactions between cells and organisms have recently become a subject of increasing attention. In this work the mechanism of formation of cell membrane nanovesicles (CNVs) was studied experimentally and theoretically. CNVs were isolated by centrifugation and washing of blood cells and observed by optical microscopy and scanning electron microscopy. The shape of the biological membrane in the budding process, as observed in phospholipid vesicles, in erythrocytes and in CNVs, was described by an unifying model. Taking the mean curvature h and the curvature deviator d of the membrane surface as the relevant parameters, the shape and the distribution of membrane constituents were determined theoretically by minimization of membrane free energy. Considering these results and previous results on vesiculation of red blood cells it was interpreted that the budding processes may lead to formation of different types of CNVs as regards the compartment (exo/endovesicles), shape (spherical/tubular/torocytic) and composition (enriched/depleted in particular kinds of molecules). It was concluded that the specificity of pinched off nanovesicles derives from the shape of the membrane constituents and not primarily from their chemical identity, which explains evidences on great heterogeneity of isolated extracellular vesicles with respect to composition.

自動血球洗浄装置「セルウォッシャーUltraCW II」の基礎的性能評価

自動血球洗浄装置「セルウォッシャーUltraCW II」の基礎的性能評価

The association between high sensitivity C-reactive protein and micronutrient levels: A cross-sectional analysis based on a laboratory database

Low-grade chronic inflammation is a condition underlying many serious diseases but there is no good single biomarker which can estimate and monitor the severity of the inflammation. C-reactive protein (CRP) is the best validated and most extensively used marker. The aims of the study were to investigate the extent to which CRP levels associate with levels of micronutrients. We retrieved the levels of S-hsCRP and nutritional variables fB-β-carotenes, fS-Q10 (Ubiquinon), fS-Fe, E-Cu, fS-A vitamin, B-Se, B-Zn, and fB-B12 vitamin from the database of clinical laboratory Mila Oy from the years 1988-2018, a total of nearly 18800 samples from outpatient clinics, Helsinki and Oulu, Finland. Sample sizes for nutritional variables measured concurrently with S-hsCRP varied between 4356 and 8621. S-hsCRP levels were categorized into five ordered categories. The levels of each micronutrient in those categories were compared using analysis of variance (ANOVA). Males and females were analyzed separately. It was observed that an increase of S-hsCRP associated with the decrease of fS-Fe (p<0.001 for both genders); fS-A vitamin (p<0.001 for both genders), and fS-β-carotenes (p<0.001 for both genders); these are considered negative acute phase reactants. For both genders there was no significant association between the levels of fS-B12 vitamin (p=0.14 for males; p=0.03 for females), fS-Q10 (p<0.001 for males; p=0.06 for females) and fB-Se (p<0.001 for males; p=0.01 for females) and the categorized S-hsCRP. In contrast, fB-Zn (p<0.001 for both genders) behaved like a positive acute phase reactant whereas copper measured from washed blood cells (E-Cu) did not display any significant associations with S-hsCRP (p=0.001 for males; p=0.05 for females). A linear association was observed for some micronutrients - the higher the degree of low-grade inflammation (S-hsCRP), the more disturbed were the levels of some micronutrients. For clinicians, this finding means that inflammation needs to be acknowledged when assessing micronutrient deficiency. Substitution therapy should be implemented only after the inflammation has been rectified.

Distinct effects of daratumumab on indirect and direct antiglobulin tests: a new method employing 0.01 mol/L dithiothreitol for negating the daratumumab interference with preserving K antigenicity (Osaka method)

There is an increasing demand for daratumumab (DARA), an immunoglobulin (Ig)G1κ monoclonal antibody (MoAb) that recognizes CD38, to manage relapsed or refractory multiple myeloma (MM) patients. However, DARA leads to positive and panreactive agglutination reactions in indirect antiglobulin tests (IATs) in vitro (the DARA interference). In addition, effects of DARA on red blood cells (RBCs) in vivo remains elusive. To develop a new method to negate the DARA interference, the effects of various concentrations of dithiothreitol (DTT) on RBC CD38 and Kell antigenicity in combination with an automatic blood cell washing centrifuge were compared with the AABB standard procedure in parallel. Moreover, direct antiglobulin tests (DATs) for RBCs in DARA-treated MM patients were examined. A quantity of 0.01 mol/L DTT as well as the AABB procedure (equivalent to 0.15 mol/L DTT in our procedure) markedly reduced the reactivity of phycoerythrin-mouse anti-CD38 MoAb and DARA with RBCs. In sharp contrast to the AABB procedure, 0.01 mol/L DTT partially preserved K antigenicity and allowed the determination of phenotype of K antigen even in the presence of the DARA interference. In contrast, DAT for RBCs obtained from MM patients showed a weak positive or negative reaction. Immunoblotting further indicated that DARA induced loss of CD38 in vivo. A simple and reliable method to negate the DARA interference with partially preserving Kell antigenicity is proposed (Osaka method). CD38 antigenicity is susceptible to 0.01 mol/L DTT treatment even in the presence of DARA. Our data also demonstrate distinct effects of DARA on IAT in vitro and DAT in vivo.

Comparison of stored red blood cell washing techniques for priming extracorporeal circuits.

The aim of this study was to compare three different blood washing techniques and describe the differences for the composition of the washed red blood cells (RBC). Stored RBCs less than 5 days old were washed using three different techniques. 1) Washing with normal saline with the COBE Model 2991 blood processor in the blood bank (BB-S). 2) Washing with normal saline with the Continuous AutoTransfusion System (C.A.T.S) in the operating room (OR-S). 3) Washing with Plasma-Lyte with the C.A.T.S in the operating room (OR-PL). Then, we compared the values for hemoglobin (Hb), hematocrit (Hct), blood volume, RBC volume, lactate, glucose, sodium and potassium of the three different groups. Forty-five units of RBCs were washed and analyzed (15 for each technique). The OR-S RBCs, when compared to the BB-S RBCs, had lower hemoglobin (g/dL) (22.8 vs 24.1, p=0.006), lower hematocrit (%) (67 vs 71, p=0.006), higher RBC volume (ml) (161 vs 130, p<0.001), higher glucose (mg/dL) (185 vs 46, p<0.001) and lower sodium (mmol/L) (153 vs 158, p<0.001). When compared to the OR-S RBCs, the OR-PL RBCs showed higher potassium (mmol/L) (5.3 vs 2, p<0.001) and lower sodium (mmol/L) (129 vs 153, p<0.001). RBCs washed with an autotransfusion device had a higher RBC volume and more physiological levels of glucose and sodium when compared with the blood processor in the blood bank. It can be an alternative option to use RBCs washed with an autotransfusion device for priming the extracorporeal circuits utilized in patients undergoing cardiac surgery.

Randomized trial of red cell washing for the prevention of transfusion-associated organ injury in cardiac surgery

BackgroundExperimental studies suggest that mechanical cell washing to remove pro-inflammatory components that accumulate in the supernatant of stored donor red blood cells (RBCs) might reduce inflammation and organ injury in transfused patients. MethodsCardiac surgery patients at increased risk of large-volume RBC transfusion were eligible. Participants were randomized to receive either mechanically washed allogenic RBCs or standard care RBCs. The primary outcome was serum interleukin-8 measured at baseline and at four postsurgery time points. A mechanism substudy evaluated the effects of washing on stored RBCs in vitro and on markers of platelet, leucocyte, and endothelial activation in trial subjects. ResultsSixty adult cardiac surgery patients at three UK cardiac centres were enrolled between September 2013 and March 2015. Subjects received a median of 3.5 (interquartile range 2–5.5) RBC units, stored for a mean of 21 (sd 5.2) days, within 48 h of surgery. Mechanical washing reduced concentrations of RBC-derived microvesicles but increased cell-free haemoglobin concentrations in RBC supernatant relative to standard care RBC supernatant. There was no difference between groups with respect to perioperative serum interleukin-8 values [adjusted mean difference 0.239 (95% confidence intervals −0.231, 0.709), P=0.318] or concentrations of plasma RBC microvesicles, platelet and leucocyte activation, plasma cell-free haemoglobin, endothelial activation, or biomarkers of heart, lung, or kidney injury. ConclusionsThese results do not support a hypothesis that allogenic red blood cell washing has clinical benefits in cardiac surgery. Clinical trial registrationISRCTN 27076315.

Abstract 19379: Red Blood Cell Washing Increases Kidney Injury in Cardiac Surgery Patients - The Role of Oxidised Lipids and Oxidative Stress

Introduction: Allogeneic red blood cell (RBC) transfusion in cardiac surgery patients increases the risk of pulmonary and renal morbidity. These associations are attributable to the ‘storage lesion’ - accumulation of metabolites in the RBC supernatant that includes pro-inflammatory lipids, cell-free hemoglobin and energy depletion. Hypothesis: Storage lesion components trigger different events that lead to organ injury. Reducing storage lesion by RBC washing or rejuvenation will reduce organ injury. Methods: Samples from 60 cardiac patients participating in a randomized controlled trial of RBC washing prior to transfusion versus standard care were analysed for inflammatory markers. In a complementary experiment, 4 groups of pigs received crystalloid infusion or 14-day old RBC that were left untreated, washed or rejuvenated (Rejuvesol, Citra Labs, IN). Inflammatory markers, free hemoglobin and iron were measured in blood. Leukocyte invasion was determined by immunohistochemistry. The results were verified and further analysed in an in vitro inflammatory model. Results: Transfusion of stored RBC (~22 days) into humans or pigs was associated with leukocyte activation, and attenuated by washing. That was attributed to oxidised lipids on transfused RBC derived microvesicles that activated monocytes and platelets, as revealed in depletion experiments. Unexpectedly, transfusion of washed RBC increased kidney injury, which correlated with higher free haemoglobin levels, oxidative stress (confirmed by HMOX-1 levels and protein carbonylation), and endothelial injury in washed RBC recipients. Oxidative stress triggered non-standard endothelial activation defined by expression of VLA5 and fibronectin retention. Either pathway was reduced when rejuvenated blood was transfused. Conclusion: Monocytes/platelets and endothelium are activated by oxidized lipids/MV and reactive free haem, respectively, and can be alleviated by RBC rejuvenation.

Introduction of a closed-system cell processor for red blood cell washing: postimplementation monitoring of safety and efficacy.

After introduction of a closed-system cell processor, the effect of this product change on safety, efficacy, and utilization of washed red blood cells (RBCs) was assessed. This study was a pre-/postimplementation observational study. Efficacy data were collected from sequentially transfused washed RBCs received as prophylactic therapy by β-thalassemia patients during a 3-month period before and after implementation of the Haemonetics ACP215 closed-system processor. Before implementation, an open system (TerumoBCT COBE 2991) was used to wash RBCs. The primary endpoint for efficacy was a change in hemoglobin (Hb) concentration corrected for the duration between transfusions. The primary endpoint for safety was the frequency of adverse transfusion reactions (ATRs) in all washed RBCs provided by Canadian Blood Services to the transfusion service for 12 months before and after implementation. Data were analyzed from more than 300 RBCs transfused to 31 recipients before implementation and 29 recipients after implementation. The number of units transfused per episode reduced significantly after implementation, from a mean of 3.5 units to a mean of 3.1 units (p<0.005). The corrected change in Hb concentration was not significantly different before and after implementation. ATRs occurred in 0.15% of transfusions both before and after implementation. Safety and efficacy of washed RBCs were not affected after introduction of a closed-system cell processor. The ACP215 allowed for an extended expiry time, improving inventory management and overall utilization of washed RBCs. Transfusion of fewer RBCs per episode reduced exposure of recipients to allogeneic blood products while maintaining efficacy.

Red blood cell washing, nitrite therapy, and antiheme therapies prevent stored red blood cell toxicity after trauma–hemorrhage

Transfusion of stored red blood cells (RBCs) is associated with increased morbidity and mortality in trauma patients. Pro-oxidant, pro-inflammatory, and nitric oxide (NO) scavenging properties of stored RBCs are thought to underlie this association. In this study we determined the effects of RBC washing and nitrite and antiheme therapy on stored RBC-dependent toxicity in the setting of trauma-induced hemorrhage. A murine (C57BL/6) model of trauma–hemorrhage and resuscitation with 1 or 3 units of RBCs stored for 0–10 days was used. Tested variables included washing RBCs to remove lower MW components that scavenge NO, NO-repletion therapy using nitrite, or mitigation of free heme toxicity by heme scavenging or preventing TLR4 activation. Stored RBC toxicity was determined by assessment of acute lung injury indices (airway edema and inflammation) and survival. Transfusion with 5 day RBCs increased acute lung injury indexed by BAL protein and neutrophil accumulation. Washing 5 day RBCs prior to transfusion did not decrease this injury, whereas nitrite therapy did. Transfusion with 10 day RBCs elicited a more severe injury resulting in ~90% lethality, compared to <15% with 5 day RBCs. Both washing and nitrite therapy significantly protected against 10 day RBC-induced lethality, suggesting that washing may be protective when the injury stimulus is more severe. Finally, a spectral deconvolution assay was developed to simultaneously measure free heme and hemoglobin in stored RBC supernatants, which demonstrated significant increases of both in stored human and mouse RBCs. Transfusion with free heme partially recapitulated the toxicity mediated by stored RBCs. Furthermore, inhibition of TLR4 signaling, which is stimulated by heme, using TAK-242, or hemopexin-dependent sequestration of free heme significantly protected against both 5 day and 10 day mouse RBC-dependent toxicity. These data suggest that RBC washing, nitrite therapy, and/or antiheme and TLR4 strategies may prevent stored RBC toxicities.

High Shear Dependent von Willebrand Factor Self-assembly Fostered by Platelet Interaction and Controlled by ADAMTS13

IntroductionThe paradigm of activation induced platelet aggregation has recently been refuted under blood flow conditions with shear rates exceeding 20,000s-1. These lead to reversible rolling platelet aggregates, which were dependent on the presence of immobilized and soluble von Willebrand factor. Material and MethodsIn vitro experiments using direct fluorescence video-microscopy were performed in wall parallel and stagnation point flow chambers with shear rates raised from 20,000 to 50,000s-1. Washed blood cell suspension containing recombinant von Willebrand factor (rVWF) was perfused over rVWF or collagen coated surfaces. ResultsHere we show for the first time with the visualization of rVWF that not only colloid and polymer, i.e. platelets and VWF, form a composite, but that VWF itself is capable of entirely reversible self-assembly. On a collagen surface the platelet-VWF-conglomerates did not roll but VWF nets bound permanently to the collagen fibers and captured and immobilized platelets from the flow. Lowering the shear rate below the threshold of 20,000s-1 no longer dissolved these deposits. Ultralarge multimer containing rVWF was most effective compared to normal sized rVWF. The presence of ADAMTS13 limited rolling aggregate and platelet-VWF-conglomerate formation to a time window of 7-8minutes. Changing wall parallel flow to stagnation point flow halved the required shear rate threshold. ConclusionWe conclude that flow dynamics can trigger reversible von Willebrand factor self-assembly and platelet-VWF-conglomerate accrual, which are regulated by ADAMTS13 to a time span needed by coagulation to stabilize it, e.g. in case of vessel injury.

Russian Pioneers of Therapeutic Hemapheresis and Extracorporeal Hemocorrection: 100‐Year Anniversary of the World's First Successful Plasmapheresis

On 15 February 2013 (2 February on the Julian Calendar) we celebrated the 100-year anniversary of the world's first successful experimental plasmapheresis. Scientific research projects in this field were carried out by the Department of Infectious Disease, Russian Imperial Medical Surgical Academy located in Saint-Petersburg. Doctor of Medical Sciences and Professor Vadim A. Yurevich was a Principal Investigator for this research, which in 1913 resulted in the discovery of a new way of treatment. The results were published in Russki Vratch (Russian Physician) Journal no. 18 (1914) - V.A. Yurevich and N.K. Rosenberg "For the Question Regarding Washing of Blood Outside of the Body and the Vitality of Red Blood Cells". There was no terminology offered for this medical innovation at that time. Plasma removal was performed not solely, but in combination with washing of blood cells returned to the patient. Nowadays this combination is still considered to be more effective than separate plasmapheresis. According to the published experimental protocols this new treatment was done on 15 February (2 February on the Julian Calendar or "old style"). One year later in 1914 a famous researcher, John Abel and coauthors, repeated a separate plasma removal treatment with retransfusion of the blood cells and suggested the term "plasmapheresis", which is now official. The article entitled "Plasma Removal With Return of Corpuscles (Plasmapheresis)", written by Abel was published 3 months later than the article by Professor Yurevich. In 1924, Dr Ivan P. Mikhailovskiy repeated experiments by Yurevich and Rosenberg in vivo on a dog model, confirmed the clinical efficiency and developed the methodology in his article "Washing of Blood In Vivo, the Methodology, Problems, and Importance for the Treatment of Toxic Conditions."

Abstract A204: Flow cytometric assay of proteasome inhibition in whole blood.

Abstract Introduction: Two proteasome inhibitors are currently approved by the US FDA for the treatment of multiple myeloma: bortezomib and carfilzomib. Both drugs make a covalent bond with a Thr in the active site of the 20S catalytic β5 subunit. While the covalent bond is hydrolyzable in the case of bortezomib, its binding is characterized by a long residency time with a half-life of close to 2 hours. The current clinical surrogate assay measures the suppression of proteasome activity in pooled cell lysates from patients’ blood or tissue following the administration of inhibitor. As such, this assay depends upon the long residency time of current inhibitors. Second-generation proteasome inhibitors that are in development, such as ixazomib, have a much faster off-rate, making accurate measurement of inhibition in diluted cell lysates problematic. Our objective was to develop a surrogate proteasome assay for use in whole blood, without requiring cell lysis, thus preventing sample dilution. Methods: Whole-blood samples were pre-incubated with proteasome inhibitors for 1-2 hours at 37°C. Subsequently, samples were stimulated with LPS at 37°C, then fixed and permeabilized using PerFix-P (Beckman Coulter). Washed white blood cells were stained with IκBα-AF488 (Cell Signaling Technologies), CD45-KrO, and CD14-PC7 monoclonal antibodies (Beckman Coulter) and analyzed using a Gallios™ flow cytometer. Results and Discussion: During the classical activation of NF-κB signaling, the NF-κB inhibitor, IκBα, undergoes phosphorylation and ubiquitination, followed by rapid proteasomal degradation. Released NF-κB complexes can then translocate to the nucleus, where they initiate transcriptional activation. In this study, we analyzed the LPS-induced degradation of IκBα in whole-blood monocytes by flow cytometry. We found a 70-80% decrease in the median fluorescent intensity (MFI) of IκBα-AF488 after 10 min of LPS stimulation. The kinetic profile and magnitude of the MFI reduction were highly reproducible in whole-blood monocytes from healthy donors, as well as in a limited number of blood samples obtained from multiple-myeloma patients. The MFI reduction was inhibited in a dose-dependent manner by different proteasome inhibitors, including bortezomib, carfilzomib, and MG132. To increase the specificity of the assay, ERK phosphorylation can also be monitored simultaneously within the same cells. ERK phosphorylation in whole-blood monocytes peaks at ∼10 min after LPS stimulation, and is caused by the release of TPL2 from its complex with p105 (unprocessed NF-κB1) following the proteasomal processing of p105 into p50. Because of this, ERK phosphorylation is also inhibited by proteasome inhibitors in a dose-dependent manner, similar to IκBα. This assay can be optimized for different cell types within whole blood by using different activators, although the kinetics of IκBα degradation varies. Conclusions: We have established a fast and convenient assay to measure proteasome activity and inhibition in whole blood leukocytes. This assay can be used for investigation of the PK/PD relationships of new-generation proteasome inhibitors with fast off-rates. Disclaimer: PerFix-P and Gallios™ are for research use only. Not for use in diagnostic procedures. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A204. Citation Format: Sergei Gulnik, T. Vincent Shankey, Lidice L. Lopez, David W. Hedley, Sue Chow. Flow cytometric assay of proteasome inhibition in whole blood. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A204.

Erythrocyte Adhesion in Hemodialysis Patients – A Novel Potential Contributor to Inflammation in End-Stage Renal Disease.

AbstractAbstract 2092 Background:Widespread adhesion of erythrocytes in the vasculature would be incompatible with life. However, in illnesses such as sickle cell disease, malaria and diabetes, adhesive RBCs have been documented and are likely contributors to disease severity, inflammation and coagulation activation. End-stage renal disease is characterized by profound, global inflammation and relative thrombophilia. While we, and others, have reported the extensive exposure of RBC phosphatidylserine in patients on hemodialysis, the role of RBC adhesion in inflammation has never been examined in this condition. We performed an analysis of RBCs from hemodialysis patients to characterize their ability to adhere to other cells, elucidate the potential mechanisms of adhesion, and to relate RBC adhesion to the inflammatory state of hemodialysis. Methods:This study was conducted in a cohort of 20 African American patients receiving in-center hemodialysis at 2 separate dialysis clinics as part of a study of sickle cell trait in hemodialysis. There were 9 of 20 patients with documented sickle cell trait. Blood samples were drawn immediately pre-dialysis and prior to administration of heparin. Plasma samples were processed to minimize platelet activation, aliquoted, and frozen for batch analysis. Plasma factors (RANTES, CRP & CD40L) were analyzed via ELISA. Adhesion events in whole blood were detected via two color flow cytometry. Cause of renal failure, mode of venous access and relevant clinical data were obtained from patient charts. All adhesion assays with washed blood cells were conducted under static conditions using microvascular endothelial cells. All studies were compared to those results of healthy control patients (n =11). Spearman’s regression analysis was performed to analyze the correlation between continuous variables. Mann-Whitney U and Kruskal Wallis tests were used to compare continuous variables between groups. A p<0.05 was considered significant. Results:We found that RBCs from patients on hemodialysis were significantly more adhesive than those from healthy controls. In whole blood, we detected marked RBC adhesion to T-cells (median % of T-cells bound to RBCs: 67.9 [hemodialysis] vs. 10.55 [healthy control]) and platelets (median % of platelets bound to RBCs: 33.0 vs. 1.1%). We also noted significant RBC adhesion to neutrophils (median % of neutrophils bound to RBCs: 11.0% vs. 0.1%). Incubation of healthy RBCs with plasma from hemodialysis patients, but not healthy control plasma, was sufficient to induce RBC adhesion to cultured endothelial cells (median RBCs/mm2: 8.0 vs. 0.5) and to T-cells (median % T-cells bound to RBCs: 30.0 vs. 5.0). Plasma from hemodialysis patients also induced phosphatidylserine exposure on healthy RBCs. Phosphatidylserine exposure on the RBC appeared to mediate RBC adhesion to endothelial cells as annexin V significantly reduced RBC adherence (8.0 vs 3.8 RBC/mm2). The extent to which RBCs in hemodialysis patients adhered to T-cells directly correlated with both plasma RANTES (rspearman = 0.65, 95% CI: 0.247–0.864) and CD40L levels (rspearman= 0.60, 95% CI: 0.1645 – 0.847), but not plasma CRP levels. There was also no significant difference in adhesion of RBCs due to cause of renal failure (diabetes, hypertension, or glomerulonephritis) or presence of sickle cell trait. Conclusions:We describe for the first time a novel adhesion of RBCs in patients receiving hemodialysis and how this adhesion may relate to inflammation in these patients. Our findings also suggest that factors in uremic plasma are sufficient to induce phosphatidylserine exposure on RBCs. This exposure, in addition to providing a site for thrombin generation, also serves as an adhesive moiety on RBCs for endothelial cells. These data may describe an unrecognized etiology of inflammation in hemodialysis and end-stage renal disease. Disclosures:No relevant conflicts of interest to declare.

Methadone concentrations in blood, plasma, and oral fluid determined by isotope-dilution gas chromatography–mass spectrometry

Methadone (MTD) is widely used for detoxification of heroin addicts and also in pain management programs. Information about the distribution of methadone between blood, plasma, and alternative specimens, such as oral fluid (OF), is needed in clinical, forensic, and traffic medicine when analytical results are interpreted. We determined MTD and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in blood, plasma, blood cells, and OF by gas chromatography-mass spectrometry (GC-MS) after adding deuterium-labeled internal standards. The analytical limits of quantitation for MTD and EDDP by this method were 20 and 3 ng/mL, respectively. The amounts of MTD and EDDP were higher in plasma (80.4 % and 76.5 %) compared with blood cells (19.6 % and 23.5 %) and we found that repeated washing of blood cells with phosphate-buffered saline increased the amounts in plasma (93.6 % and 88.6 %). Mean plasma/blood concentration ratios of MTD and EDDP in spiked samples (N = 5) were 1.27 and 1.21, respectively. In clinical samples from patients (N = 46), the concentrations of MTD in plasma and whole blood were highly correlated (r = 0.92, p < 0.001) and mean (median) plasma/blood distribution ratios were 1.43 (1.41). The correlations between MTD in OF and plasma (r = 0.46) and OF and blood (r = 0.52) were also statistically significant (p < 0.001) and the mean OF/plasma and OF/blood distribution ratios were 0.55 and 0.77, respectively. The MTD concentration in OF decreased as salivary pH increased (more basic). These results will prove useful in clinical and forensic medicine when MTD concentrations in alternative specimens are compared and contrasted.

Shear Induced Reversible Conglomerates of Von Willebrand Factor Networks with Aggregates of Inactivated Platelets - a Fast Switchable Plugging Mechanism

AbstractAbstract 2104Thrombus growth rate has been reported to be a linear function of very high shear rates beyond 40,000 s-1 (Ku DN, Bioreology. 2007;44(4):273-84), which are thought to occur in severe arterial stenosis. We therefore applied shear rates continuously increasing from 2,000 s-1 to 40,000 s-1 and examined platelet and von Willebrand factor (VWF) interaction in a parallel plate flow chamber under direct visualization. Above a critical threshold of 10,000 to 15,000 s-1 aggregates of inactivated platelets formed when VWF was present in solution and immobilized on the perfused surface (Ruggeri, … Reininger; Blood, 108: 1903–1910, 2006). A new finding was the shear dependent simultaneous formation of VWF strands in and around those platelet aggregates, growing to strands up to several microns thick and up to several tens of microns long, thus forming large VWF networks. Platelets appeared to be enmeshed in the VWF networks but were nevertheless a prerequsite for their formation, mutually fostering each other. Platelet-VWF conglomerates formed at the perfused VWF surface, were constantly rolling in the flow direction, grew in size with increasing shear rate, and were completely reversible below the critical shear rate threshold of 10,000 s-1. VWF networks assembled in whole blood as well as in washed blood cell suspensions reconstituted with VWF. Recombinant full length VWF generated larger VWF networks than a commercial VWF preparation from pooled human plasma. Perfusion of collagen coated surfaces caused assembly of VWF networks anchored to the collagen fibrils and capture of activation-blocked platelets within them at shear rates of 2,000 s-1, leading to residence times of platelet-VWF conglomerates of more than a minute. Newly formed platelet conglomerates could be visualized rolling on the surfaces of already attached conglomerates. When we used a gain of function mutation of VWF, rolling platelet aggregates could be detected at a shear rate as low as 1,500 s-1. We conclude that shear rate activation of VWF leads to reversible platelet-VWF network generation, which may be a crucial mechanism of fast platelet accrual and prolonged arrest times at sites of thrombosis as well as physiological hemostasis. Disclosures:No relevant conflicts of interest to declare.

Woundstat™ Topical Hemostat Reverses Clotting Diatheses in Models of Acquired and Congenital Hemophilia.

Globally over 200,000 persons are afflicted with hemophilia. Topical treatments for perioperative intervention of hemophiliac bleeding are currently limited to fibrin glues, which have been used successfully to carry out dental extractions, orthopedic and non-orthopedic surgeries, and for circumcisions. Drawbacks of fibrin glues include risk of blood borne pathogen contamination and immunologic cross-reaction to factors II and V. Although topical hemostatic agents are available for treating traumatic bleeding in the field by military and EMS first responders, their effectiveness for treating bleeding in hemophilia, platelet disorders, surgery; and for other hemostatic defects observed in cirrhosis and other liver diseases and during anticoagulation therapy overdose, have not been studied. A new granular smectite hemostatic agent (WoundStat™ [WS]) has demonstrated 100% efficacy in producing hemostasis during high pressure traumatic arterial bleeding (Ward et al., J Trauma , 63:276–284, 2007; Carraway et al., Resuscitation , 78:230–235, 2008) and has been cleared for external use in cases of moderate to severe bleeds. Here, we evaluated the effectiveness of WS in models of acquired and congenital hemophilia using a modified whole blood recalcified clotting time assay on the Actalyke Mini II activated clotting time (ACT) analyzer and empty G-ACT tubes devoid of contact phase activating reagents. Whole citrated blood recalcified to 10 mM with CaCl2 yielded clot times (CT) of 220 s, 210 s, and 230 s, respectively, for three normal donors. Acquired hemophilia A and B bloods were prepared by incubating normal citrated blood from an individual subject with function-blocking affinity purified polyclonal antibodies to factors VIII and IX, respectively. At near saturation, antibody to FIX (150 μg/ml) increased clotting times from 213 ±3 s to 405±11 s, while antibodies to FVIII (50 μg/ml) increased CT from 229 ± 6 s to 436 ± 17 s. Matched antibody controls to prothrombin fragment 1+2 (200 μg/ml) did not prolong baseline clot time (242 ± 5 s). Addition of WS at 1 mg/ml, shortened the prolonged clotting times of the acquired hemophilia bloods from 436 ± 17 sec to 223 ±2 sec (p< 10−4) and from 405 ±11 s to 204 ± 5 s (p< 10−4) for the hemophilia A and B bloods, respectively. Notably no differences were observed between CT of normal blood relative to acquired hemophilia bloods treated with WS (p < 0.3). Corresponding normal control blood clotting times were shortened by 1 mg/ml WS to 92 ± 8 s. To simulate classic hemophilia, plasma derived from hemophilia A and B bloods containing < 1% normal clotting activity or autologous plasma were mixed with fresh total washed blood cells from a type O subject at 40% Hematocrit. Recalcified CT for the autologous, hemophilia A and hemophilia B bloods were 251 ±5, 596 ± 36 and 370 ± 36 s, respectively. The addition of 3 mg/ml WS normalized CT of the congenital hemophilia A (183 ± 2 s) and B (245 ± 2) bloods and was procoagulant of autologous control blood (92 ± 7 s). In contrast to WS, CELOX chitosan hemostatic granules (1–3 mg/ml) did not accelerate clotting of acquired (444 ± 43 s) or congenital (508 ± 23 s) hemophilia A; acquired (474 ± 62 s) or congenital (643± 20 s) hemophilia B or normal autologous bloods (345 ± 32 s). These results suggest that WS may be a viable, cost effective, and safe treatment for hemophiliac bleeds during traumatic injury prior to emergent care or surgical intervention. Further animal and laboratory studies are required before WS can be shown to be effective during moderate to severe bleeding in hemophilia and other coagulopathies such as VWD or patients administered anticoagulant therapy.

Blood collection from the American Horseshoe Crab, Limulus polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and α2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.