Clotting factor activity in fresh frozen plasma after thawing with a new radio wave thawing device.

BACKGROUND: Massive hemorrhage usually results in rapid need of blood products. Patients in need of fresh frozen plasma (FFP) might benefit from shorter thawing times using a novel radio wave device. So far, only one study on the prototype has been published. Activities of clotting factors after thawing FFP with the radio wave device and with a system using water cushions were compared. This is the first study analyzing the quality of FFP using the fully developed radio wave thawing device UFT100. STUDY DESIGN AND METHODS: Thirty FFP units were thawed with the UFT100 and the Plasmatherm machine. Various clotting factors and inhibitors were analyzed before freezing, immediately after thawing, and after a 48-hour storage period at +4°C. RESULTS: After thawing, all factor activities were within normal ranges regardless of the thawing procedure. We observed significant differences in nearly all clotting factor activities regarding time as effector, whereas thawing with the Plasmatherm machine led to a significant decrease (>10%) only in factor V activity compared to the UFT100. CONCLUSIONS: Immediately after rapid thawing with the UFT100, all FFP units contained adequate coagulation factor activities to maintain hemostatic activity. The UFT100 does not deteriorate FFP quality compared to a conventional system. Regardless of the thawing system, the postthaw refrigerated storage caused a decrease in several clotting factors and inhibitors (factors V, VIII, and IX; von Willebrand factor activity; protein S and C activity) and a significant increase of factor XI.

First comparative analysis concerning the plasma platelet contamination during MNC collection.

BACKGROUND AND OBJECTIVES: Monocytes can be cultured into dendritic cells with addition of autologous plasma, which is highly prone to platelet contamination due to the apheresis process. Since platelets affect the maturation process of monocytes into dendritic cells and might even lead to a diminished harvest of dendritic cells, it is very important to reduce the platelet contamination. A new collection device (Spectra Optia) was analyzed, compared to two established devices (COM.TEC, Cobe Spectra) and evaluated regarding the potential generation of source plasma. MATERIALS AND METHODS: Concurrent plasma collected during leukapheresis was analyzed for residual cell contamination in a prospective study with the new Spectra Optia apheresis device (n=24) and was compared with COM.TEC and Cobe Spectra data (retrospective analysis, n=72). Donor pre-donation counts of platelets were analyzed for their predictive value of contaminating PLTs in plasma harvests. RESULTS: The newest apheresis device showed the lowest residual platelet count of the collected concurrent plasma (median 3.50×109/l) independent of pre-donation counts. The other two devices and sets had a higher platelet contamination. The contamination of the plasma with leukocytes was very low (only 2.0% were higher than 0.5×109/l). CONCLUSIONS: This study showed a significant reduction of platelet contamination of the concurrent plasma collected with the new Spectra Optia device. This plasma product with low residual platelets and leukocytes might also be used as plasma for fractionation.

Comparison of Six Different Cryoprotective Agents Used for Deep Freezing and Storage of CD34+ Cells Derived from Cord Blood and Peripheral Blood Stem Cell Concentrates.

BACKGROUND: For cryopreservation of stem cells, a cryoprotective agent is essential. Dimethyl sulfoxide is commonly used, but has deleterious effects on cell vitality in the warmth and for the recipients of stem cells. The aim of the study was to reduce DMSO and find one cryoprotective solution suitable for stem cells of different origin. Materials: Small volumes of both stem cell apheresis products and cord blood derived stem cells were frozen using six different cryoprotective solutions. Suitability of these solutions was tested by comparing cell vitalities and recovery rates of CD45 and CD34 positive cells and colony forming unit recovery rates. RESULTS: No single cryoprotective solution being significantly superior regarding all cell qualities and recovery rates could be identified. However, mixing approximately 5% DMSO with hydroxyethyl starch with a molecular weight of 450,000 Dalton showed better results for most qualities examined than DMSO alone, especially when looking at cord blood derived stem cells. CONCLUSIONS: There might not be an all-in-one cryoprotective solution suitable for every purpose regarding the cryopreservation of stem cell concentrates produced from different cell sources. However, when trying to reduce the DMSO amount used, hydroxyethyl starch of a molecular weight of 450,000 Dalton is a suitable option.

Massive intoxication with rivaroxaban, phenprocoumon, and diclofenac: A case report.

RATIONALE: Oral anticoagulants and painkillers, some with an additional effect on the coagulation system, are widely used and are therefore prone to abuse and (intentional) overdose. We report the case of a patient with a massive mixed anticoagulant intoxication. PATIENT CONCERNS: The patient had ingested 1960 mg rivaroxaban, 31.5 mg phenprocoumon, 1425 mg diclofenac, and 21,000 mg metamizole in suicidal intention. DIAGNOSES: Massive mixed anticoagulant overdose. INTERVENTIONS: The patient was closely monitored. The phenprocoumon overdose was treated by the administration of vitamin K and PCC. OUTCOMES: Despite the massive inhibition of the coagulation system, the patient did not experience bleeding apart from a slight gross hematuria. LESSONS: Despite the ingestion of a massive amount of rivaroxaban, the plasma levels were not as high as feared, due to the ceiling effect of rivaroxaban absorption. Elimination occurred according to the half-life of rivaroxaban and was not unduly prolonged by the ingested quantity.

Quality of Clotting Factor Activity in Fresh Frozen Plasma at Thaw with a Microwave System and after Storage at 4 degrees C for 48 Hours.

BACKGROUND: Uncontrolled hemorrhage in polytrauma patients usually results in rapid need of blood products. Despite the shorter thawing times of microwave devices for heating fresh frozen plasma (FFP), their use has remained controversial, and just a few laboratory analyses have been published on this topic. The aim of this study was to analyse the quality of clotting factors immediately after thawing FFP with a microwave device and after 48-hour post thaw storage at 4 degrees C. METHODS: 24 FFP units of all four ABO blood groups (six of each blood group) were thawed with a Transfusio-therm 2000 and later stored at 4 degrees C for 48 hours. Samples were drawn aseptically and investigated on various clotting factors and protein proteases (fibrinogen, antithrombin, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, vWF antigen and activity, protein S, and protein C) using standard coagulation and chromogenic assays immediately after thawing and again after a 48-hour storage period at 4 degrees C. All units were tested for both anaerobic and aerobic microbial contamination using standard operating procedures immediately after thawing. RESULTS: After thawing, all coagulation factors and protein protease activities were within normal ranges. Blood group O individuals had approximately 25% lower plasma levels of vWF antigen and activity. After a 48-hour storage period at 4 degrees C, FVIII and FIX activities declined significantly in all blood groups, whereas the remaining clotting factors remained comparably stable. CONCLUSIONS: Immediately after rapid thawing using a microwave system, all FFP units contained adequate coagulation factor activities to maintain hemostatic activity at the time of product thaw. The post thaw refrigerated storage caused an anticipated decrease in factor VIII and IX activities, but retained normal coagulation factor levels of many plasma proteins. Therefore we conclude that the Transfusio-therm 2000 has no clinically significant influence on the activity of clotting factors and plasma proteases in FFP units.

Measuring Lymphocyte Proliferation in Response to Specific Antigen and Mitogen Stimuli Using Flow Cytometry.

BACKGROUND: We present a multivariate test system using flow cytometry after in-vitro lymphocyte stimulation using 5 mitogens and 7 antigens to describe in-vitro immunofunction. METHODS: The present work is a crucial step towards establishing a simple, CFSE-based, multivariate test system that can describe the dynamics of stimulus-induced lymphocyte proliferation with considerably more precision than is possible with the radionucleotide method using 3H-thymidine. Using multicolour flow cytometry, our method allows additional phenotyping of the proliferating cells and quantifies the proliferation behaviour by precisely resolving daughter generations and determining the precursor frequency. CONCLUSIONS: Taking the calculated apoptosis parameters into account not only provides additional information about the stimulus-specific response behaviour but also improves the validity of the commonly used proliferation indices. Not only can we confirm previous findings that healthy people have marked differences in a multivariate test system in terms of the individual in-vitro reactivity to various stimuli but also substantiate that the response pattern of an individual is remarkably constant. In follow-up studies we can show for the first time that the results of immunofunctional testing do not change over a period of at least 6 months and appear to be an inherent characteristic of the individual and thus possibly have a genetic basis.

Influence of Irradiation on Leukodepleted Small Unit Red Blood Cell (RBC) Bags for Infant Transfusion in Additive Solution SAG-M.

BACKGROUND: To reduce transfusion-associated graft-versus-host disease irradiation of blood products is widely accepted. There is little data about the effect of gamma-irradiation on leukoreduced RBCs stored in SAG-M that are subdivided for use in transfusion to preterm infants and small children. METHODS: We studied 30 leukoreduced SAG-M preserved RBC bags. All RBCs were leukoreduced on the collection day. The 30 units were divided into two groups. Every unit was divided into three bags. One of these bags served as nonirradiated control (group 1A, group 2A), the other two were gamma-irradiated at different times. In vitro evaluation of irradiated and nonirradiated units was performed on the days +3, +7, +14, +21, and +28 from the day of collection. RESULTS: Gamma irradiation induced a higher increase of extracellular hemoglobin, LDH, and potassium than non-irradiated storage over the time. No irradiated or non-irradiated unit showed a hemolysis rate over the recommended limit of 0.8% over the 28 day period. CONCLUSIONS: Our findings show that subdivision of RBCs does not have an appreciable influence on the storage of leukoreduced, irradiated RBCs in AS SAG-M. Our "worst case scenario" was irradiation on day +3 after donation and subsequent storage until day +28. Especially for infant use, it is important to have the possibility to irradiate even late after donation, because this procedure offers the possibility to use one RBC bag over a longer period of time and to reduce the donor exposure for infants. Therefore, subdivided leukoreduced RBCs can be safely irradiated until day +14 and subsequently stored until day +28 after donation.

Comparison of Simultaneous CD34+ and CD3+ Quantification with a Modified Stem Cell Enumeration Kit on Two Different Flow Cytometers.

BACKGROUND: Quantification of CD34+ cells in peripheral blood stem cell apheresis is normally performed by single platform flow cytometric measurements according to the ISHAGE protocol. Peripheral blood stem cell concentrates (PBSC) produced by apheresis normally contain many T cells. Those T cells can be used for production of donor lymphocyte infusion doses, if abundant amounts of CD34+ cells have been collected. Therefore, it is of interest to know both the CD3+ and the CD34+ cell count of allogeneic PBSC. This is the first study comparing the performance of a modified ISHAGE protocol allowing additional quantification of CD3+ cells on two different flow cytometers, the FACSCalibur and the FACSVerse, respectively. METHODS: CD45+ and CD34+ cell concentrations were measured using a standard and a modified ISHAGE protocol including CD3+ cell quantification on both machines. All cell concentrations were measured using a Trucount bead based stem cell enumeration kit. The FACSVerse machine can additionally be equipped with a sample volume sensor allowing cell quantification without using beads. The samples analysed were taken from granulocyte-colony-stimulating factor mobilized peripheral blood stem cell apheresis procedures (pre- and post-apheresis, and apheresis concentrate). RESULTS: There were no significant differences in cell concentrations measured by the standard and modified ISHAGE protocol, regardless of which machine had been used when using bead quantification. No significant differences between the results of the two flow cytometers using the modified ISHAGE protocol were observed. Pearson´s correlation was always > 0.96, and regression coefficients were higher than 0.93. The only significant differences were observed between bead quantification and volume sensor quantification on the FACSVerse machine. CONCLUSIONS: The modified ISHAGE protocol can effectively be used on both flow cytometers tested, especially if bead quantification is used.

Transfusion-related alloimmune neutropenia with no pulmonary complications: one donor-five cases.

BACKGROUND: Neutrophil alloantibodies are well-known triggers of transfusion-related acute lung injury (TRALI) and also cause immune neutropenia. Alloimmune neutropenia due to transfusion is an isolated phenomenon that is only rarely identified. Its incidence is specified in the literature as being less than one in 10,000 transfused plasma-containing units. We expect that this phenomenon is underreported. STUDY DESIGN AND METHODS: We observed five cases of alloimmune neutropenia with no respiratory complications with only one case initially reported as a suspected transfusion reaction. The other four cases were detected in the course of the subsequent lookback investigation. RESULTS: The first case was reported as a potential transfusion reaction when a female patient showed a decrease in the white blood cell count after a platelet (PLT) transfusion. Examinations of the donor blood revealed an antibody against the human neutrophil antigen HNA-1b; the recipient was typed HNA-1b positive and HNA-1a negative. After examining the blood counts of other patients who previously received PLT concentrates from the same donor, we identified four other patients with an unreported decrease in the leukocyte and/or granulocyte count of more than approximately 50% after transfusion. CONCLUSION: HNA antibodies are generally regarded as potential triggers of TRALI. Here we describe an HNA antibody that reproducibly caused transfusion-related neutropenia only without pulmonary complications. Factors predisposing patients to TRALI development are widely discussed. Our case suggests that antibody characteristics are also relevant in the development of TRALI. Current measures to prevent TRALI should also prevent transfusion-related alloimmune neutropenia.

Influence of Duration and Temperature of Transport and Storage Prior to Processing on Cell Quality of Cord Blood Units--A German. Experience.

BACKGROUND: In Germany, cord blood needs to be transported to the processing facility to be processed and cryopreserved within 48 hours after collection according to national guidelines. During that time, a temperature of 22 ± 4 degrees C must be maintained. The purpose of this study was to analyse the influence of temperature during transport and storage prior to processing and cryopreservation on stem cells in 2460 both autologous and allogeneic umbilical cord blood samples. METHODS: Total and viable CD45+ cells, total and viable CD34+ cells, and mononuclear cells (MNC) of cord blood and resulting leucocyte concentrate both before and after freezing were analysed by flow cytometry. Transport protocols and the records of temperature measuring chips used in transport were evaluated in order to analyse how long each unit was exposed to which temperature ranges. RESULTS: On average, the cord blood preparations were delivered within 16.4 ± 6.3 hours. No cord blood was delivered and processed later than 48 hours after donation. Temperature of transport and storage before processing had minor but sometimes significant effects on cell viability. A temperature range of 20 - 24 degrees C showed best survival rates for CD34+ cells and highest colony forming potential. CONCLUSIONS: The temperature prior to processing has little yet sometimes significant effects on cell viability in stem cell concentrates prepared from cord blood. However, the absolute differences in cell viabilities are quite small. Therefore, the effect is clinically negligible in a range from 4 degrees C to 28 degrees C if cryopreservation is done within 48 hours.

Von Willebrand factor multimer structure is neither acutely nor chronically affected by plateletpheresis.

BACKGROUND: Plateletpheresis (PltPh) exposes the donor's blood to artificial surfaces and mechanical forces such as shear stress and centrifugation. In terms of the donor's safety and the quality of the apheresis platelet concentrate (APC), possible impairment of platelet function due to PltPh should be excluded. Von Willebrand factor (VWF) plays a pivotal role in platelet adhesion and aggregation. VWF is a multimeric protein and can be damaged by adsorption or shear stresses. It is unclear whether VWF structure could be damaged during PltPh, leading to platelet dysfunction. METHODS: We analyzed VWF antigen (VWF:Ag), ristocetin cofactor (VWF:RCo), and VWF multimer structure immediately before and after apheresis in the donor and in the APC. These parameters and factor VIII activity (FVIII:C) and closure time using PFA-100 (CT) were also analyzed in blood samples taken from new donors before the first and before subsequent donations and from long-term donors. RESULTS: During apheresis, VWF:Ag falls by about 15% but the VWF multimer structure remains unchanged. In samples taken before subsequent donations, there was a tendency of VWF:Ag and FVIII:C to increase throughout the initial donations, but no alteration of multimer structure. Long-term donors, however, show a normal VWF multimer structure and normal concentrations of VWF:Ag, VWF:RCo, and FVIII:C. In some donors with low-normal VWF:Ag and VWF:RCo, PFA-100 CT was prolonged. CONCLUSIONS: VWF multimer structure is neither acutely nor chronically affected by plateletpheresis. A decrease in VWF:Ag with no functional damage only occurs acutely and can be explained by the withdrawal of plasma and dilution with the anticoagulant ACD-A due to apheresis.

Travel habits and complications in patients treated with vitamin K antagonists: a cross sectional analysis.

BACKGROUND: Travel-related conditions have impact on the quality of oral anticoagulation therapy (OAT) with vitamin K-antagonists. No predictors for travel activity and for travel-associated haemorrhage or thromboembolic complications of patients on OAT are known. METHODS: A standardised questionnaire was sent to 2500 patients on long-term OAT in Austria, Switzerland and Germany. 997 questionnaires were received (responder rate 39.9%). Ordinal or logistic regression models with travel activity before and after onset of OAT or travel-associated haemorrhages and thromboembolic complications as outcome measures were applied. RESULTS: 43.4% changed travel habits since onset of OAT with 24.9% and 18.5% reporting decreased or increased travel activity, respectively. Long-distance worldwide before OAT or having suffered from thromboembolic complications was associated with reduced travel activity. Increased travel activity was associated with more intensive travel experience, increased duration of OAT, higher education, or performing patient self-management (PSM). Travel-associated haemorrhages or thromboembolic complications were reported by 6.5% and 0.9% of the patients, respectively. Former thromboembolic complications, former bleedings and PSM were significant predictors of travel-associated complications. CONCLUSIONS: OAT also increases travel intensity. Specific medical advice prior travelling to prevent complications should be given especially to patients with former bleedings or thromboembolic complications and to those performing PSM.

The place of new oral anticoagulants in travel medicine.

New oral anticoagulants are increasingly used instead of vitamin K antagonists or low molecular weight heparins. Hence, more individuals treated with new oral anticoagulants will seek travel medicine advice. Travel medicine experts should therefore become familiar with new oral anticoagulants and with their impact and role in travel medicine. This review summarizes pharmacological characteristics and approved indications of dabigatran, rivaroxaban and apixaban, and highlights their relevance for travellers on permanent oral anticoagulation and for the prophylaxis of travellers' thrombosis. Compared to vitamin K antagonists, the new oral anticoagulants have many advantages: they do not have interactions with food, they have lower potential for drug-drug interactions and do not require regularly performed laboratory tests. The oral administration, obviating the need to carry needles and syringes during travel may give the new oral anticoagulants a further advantage over low molecular weight heparins. Clinical experience with the new oral anticoagulants, however, is still rather limited and there is concern regarding the clinical management of patients treated with new oral anticoagulants who suffer from severe bleeding or who need urgent invasive procedures. Overall, it remains an individual decision based on a risk/benefit analysis as to whether or not patients on long-term treatment with vitamin K antagonists should be switched to new oral anticoagulants for intended travel. Further caution is also indicated so that the availability of orally administered new anticoagulants should not lead to undifferentiated and unjustified prescription of anticoagulants for the prophylaxis of traveller's thrombosis.

von Willebrand factor, clotting factors, and clotting inhibitors in apheresis platelet concentrates.

BACKGROUND: Apheresis platelet concentrates (APCs) are usually stored in citrated plasma at 22°C. The stability of coagulation proteins-von Willebrand factor (vWF), clotting factors (CFs), and their inhibitors-has often been described in association with the storage of thawed plasma. However, fewer data are available regarding changes in APCs. STUDY DESIGN AND METHODS: We measured CF activities and inhibitors in APCs on the day of manufacture (Day 0) and on Days 4, 5, and 7. vWF was determined by measuring vWF antigen (vWF:Ag) and vWF ristocetin cofactor (vWF:RCo) and by multimer analysis. RESULTS: Twenty-one PCs obtained by plateletpheresis were studied. Major changes were observed for Factor (F)VIII (37% loss of activity within 4 days), FV (20% within 4 days), and protein S (76% within 4 days). All other CF activities remained higher than 80% over the 7 days. Fibrinogen and the inhibitors antithrombin and protein C remained quite stable. FXI, FXII, and FXIII actually increased during storage (8, 11, and 12% within 4 days). vWF:Ag increased during storage of APCs by 2% per day, with a relative loss of vWF:RCo and high-molecular-weight multimers. CONCLUSION: Even after 7 days of storage at 22°C, the hemostatic potential of the plasma content in APCs was roughly preserved. The increase in FXII antigen indicates that this CF may also be stored in platelets; however, this has not yet been described.

The effect of cell concentrations from different cell populations on the viability of umbilical blood stem cells.

BACKGROUND: In addition to bone marrow or peripheral blood derived stem cells, cord blood (CB) is an alternative source for hematopoietic stem cells. This report shows the impact of higher concentrations of leukocytes, mononu- clear cells (MNCs), and CD34-positive cells on the viability of CB derived stem cells after cryopreservation. METHODS: Statistical analysis of data from 5520 CB units, prepared and cryopreserved from 2003 through 2011, was performed with appropriate software. Cell concentrations for leukocytes, platelets, red blood cells (RBCs), CD34-positive leukocytes, viable leukocytes, and MNCs were determined. The proliferation and differentiation capacity was assessed in cell culture assays. RESULTS: Content of leukocytes, CD34-positive leukocytes, and MNCs decreased after thawing. The recovery rate of colony forming units (CFUs) (29.05%) correlated significantly with leukocytes, platelets, RBCs, MNCs, CD34- positive leukocytes, and viable leukocytes. The recovery rate for erythroblasts (3.33%) correlated significantly with leukocytes, CD34-positive leukocytes, MNCs, and viable leukocytes. In the different cell concentration groups only RBCs showed a negative influence on viability. The concentrations of leukocytes, platelets, and CD34-positive leukocytes before cryopreservation correlated positively with the concentrations of leukocytes, CD34-positive leukocytes, MNCs as well as with the cell viability after thawing. CONCLUSIONS: Increased cell concentrations in CB do not limit the recovery of CD34-positive leukocytes nor the viability of leukocytes or the number of CFUs after thawing. On the contrary, CB units with high cell concentrations show a better outcome than units with low cell concentrations. Only RBCs seem to have a negative influence on CB quality.

Ex vivo expansion of hematopoietic stem- and progenitor cells from cord blood in coculture with mesenchymal stroma cells from amnion, chorion, Wharton's jelly, amniotic fluid, cord blood, and bone marrow.

In most cases, the amount of hematopoietic stem and progenitor cells (HSPCs) in a single cord blood (CB) unit is not sufficient for allogenic transplantation of adults. Therefore, two CB units are usually required. The ex vivo expansion of HSPCs from CB in coculture with mesenchymal stroma cells (MSCs) might be an alternative. It was investigated, whether bone marrow-derived MSCs, which have to be obtained in an invasive procedure, introduce a further donor and increases the risk of transmissible infectious diseases for the patient can be replaced by MSCs from amnion, chorion, Wharton's jelly, amniotic fluid, and CB, which can be isolated from placental tissue which is readily available when CB is sampled. In a two-step ex vivo coculture mononuclear cells from cryopreserved CB were cultured with different MSC-feederlayers in a medium supplemented with cytokines (stem cell factor, thrombopoietin [TPO], and granulocyte colony-stimulating factor). Expansion rates were analyzed as well, by long-term culture-initiating cell (LTC-IC) and colony-forming unit (CFU) assays, as by measuring CD34(+)- and CD45(+)-cells. Due to the comparably low number of 5×10(2) to 1×10(4) CD34(+)-cells per cm(2) MSC-monolayer, we observed comparably high expansion rates from 80 to 391,000 for CFU, 70 to 313,000 for CD34(+)-, and 200 to 352,000 for CD45(+)-cells. Expansion of LTC-IC was partly observed. Compared to the literature, we found a better expansion rate of CD34(+)-cells with MSCs from all different sources. This is probably due to the comparably low number of 5×10(2) to 1×10 CD34(+)-cells per cm(2) MSC-monolayer we used. Comparably, high expansion rates were observed from 80 to 391,000 for CFUs, 70 to 313,000 for CD34(+)-, and 200 to 352,000 for CD45(+)-cells. However, the expansion of CD34(+)-cells was significantly more effective with MSCs from bone marrow compared to MSCs from amnion, chorion, and Wharton's jelly. The comparison of MSCs from bone marrow with MSCs from CB and amniotic fluid showed no significant difference. We conclude that MSCs from placental tissues might be useful in the expansion of HSPCs, at least if low numbers of CD34(+)-cells per cm(2) MSC-monolayer and a high TPO concentration are implemented in the expansion culture.