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.

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.

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.

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.

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.

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, 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.

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.

Microparticle detection in platelet products by three different methods.

BACKGROUND: Standardization of platelet-derived microparticle (PMP) enumeration by flow cytometry (FCM) is limited due to its intrinsic characteristics. Because of high clinical relevance of microparticle (MP) detection, standardization of MP assays is required. STUDY DESIGN AND METHODS: This prospective paired study analyzed 31 healthy blood donors (18 male, 13 female) and compared pre- and postdonation results of donors with results of plateletpheresis products by three different methods. PMP counts were analyzed by FCM using calibrated beads of defined diameter and annexin V-fluorescein isothiocyanate and CD41-phycoerythrin staining. MP activity was tested by prothrombinase assay (enzyme-linked immunosorbent assay [ELISA]) and a procoagulant phospholipid-dependent clotting time assay (STA-Procoag-PPL, Diagnostica Stago S.A.S.). RESULTS: PMP concentration was more than threefold higher in single-platelet units (SPUs) and resulted in higher PMP yields in SPUs compared to double-platelet units (DPUs). The ELISA and the procoagulant clotting assay also revealed a significant higher MP activity in SPUs compared to DPUs. The results of the procoagulation clotting assay correlated inversely with PMP counts obtained by FCM (r = -0.685, p < 0.001) and with the MP activity measured by ELISA (r = -0.641, p < 0.001). CONCLUSION: Three different methods for MP detection showed good correlations of results, albeit the basis for MP analysis was different. Even if FCM is considered the "gold standard" of MP detection there are still technical limitations concerning detection of small MP. The procoagulant STA-Procoag-PPL assay and the prothrombinase ELISA assay could be useful additional MP tests. Regarding the interpretation of quantitative results of MPs, preanalytical conditions must be optimized and standardized.

Leukapheresis in non-cytokine-stimulated donors with a new apheresis system: first-time collection results and evaluation of subsequent cryopreservation.

BACKGROUND: Adoptive cell therapy based on mononuclear cells (MNCs) became an important modality of cancer immunotherapy. Data about collection results and donor response of leukapheresis with the Spectra Optia v.5.0 (Terumo BCT) in nonmobilized donors are required. STUDY DESIGN AND METHODS: Twelve MNC collections were performed using the Spectra Optia v.5.0 in non-cytokine-stimulated donors. Leukapheresis products and peripheral blood samples from donors were assayed for CD45+, CD34+, CD3+, and CD14+ cells by flow cytometry. Prefreeze and postthaw cell counts, cell viability, and numbers of colony-forming units were assessed in cryobags and compared to data from cryovials. RESULTS: Leukapheresis yielded a mean of 5.26×10(9) ±2.2×10(9) CD45+ cells, 1.5×10(9) ±0.77×10(9) CD14+ monocytes, and 2.28×10(9) ±1.2×10(9) CD3+ Tcells by processing 6690±930mL of whole blood. A significant positive correlation between yield of CD3+ Tcells and residual platelets (PLTs) and red blood cells (RBCs) was observed. This did not apply for CD34+ and CD14+ white blood cell subsets. Mean collection efficiencies for CD14+ monocytes and CD3+ Tcells were 61.8±17 and 37.2±18%, respectively. Recovery of CD14+ cells after cryopreservation was 75.2±8.2%, which was significantly lower than recovery of CD45+ cells (81.4±5.5%; p=0.01). CONCLUSION: This study of a small cohort demonstrates that the Spectra Optia v.5.0 is capable of collecting low product volumes with satisfactory MNC yields and low residual RBCs and PLTs in non-cytokine-mobilized apheresis. Our data suggest that cryovials can serve as a representative surrogate for the primary product cryobag.

A detailed examination of platelet function inhibition by nitric oxide in platelet-rich plasma and whole blood.

BACKGROUND: The question of whether novel instruments such as multiple electrode aggregometry (MEA) can be used for measurement of the effects of nitric oxide (NO) on platelets (PLTs) has not been examined. METHODS: Therefore, we compared the effects of NO concentrations (1, 10, and 100 microM) on the PLT aggregation response to ADP, arachidonic acid (AA), collagen, ristocetin, and thrombin receptor-activating peptide 6 (TRAP6) using light transmission aggregometry (LTA) and multiple electrode aggregometry (MEA) and examined the effects of NO using the platelet function analyzer (PFA)-100. RESULTS: The response of PLTs to ADP and AA was strongly inhibited by all NO concentrations in LTA and MEA. The inhibition of the responses to ristocetin and collagen was detectable in MEA at lower NO concentrations than in LTA. However, the typically increasing lag phase between collagen addition and the aggregation response in the presence of NO was more obvious in LTA. TRAP caused a reproducible early response in the presence of NO in LTA which was followed by rapid PLT disaggregation, whereas even 100 microM NO did not inhibit the response to TRAP in MEA. Finally, NO prolonged the in-vitro bleeding time remarkably more in the PFA-100 collagen-epinephrin cartridge than in the collagen-ADP cartridge. CONCLUSIONS: Whole blood versus PLT rich plasma, citrate versus hirudin, and high versus low shear influenced the effects of NO. This shows that a careful selection of models and potentially a combination of different methods is appropriate for a differentiated evaluation of pharmacological or physiological mechanisms of NO-donors or of NO-inhibitors.

Effects of immediate or delayed addition of platelet additive solution on the in vitro quality of apheresis platelets.

BACKGROUND: There is little knowledge how different hold times of hyperconcentrated platelet (PLT) suspensions (HPSs) before the addition of platelet additive solution (PAS) might affect PLT quality. We compared the in vitro quality of single-donor PLT concentrates (SDPs) with immediate or delayed PAS addition and studied the quality of collected concurrent plasma (CP). STUDY DESIGN AND METHODS: We collected 6×10(11) PLTs in 175 of mL plasma and CP from 31 donors. The HPSs were split into two parts, with 162 mL of modified PAS III (PAS-IIIM) added immediately (0hr-SDP) or 2 hours later (2hr-SDP). Final SDPs had a targeted concentration of 1.2×10(12) PLTs/L and a PAS proportion of 65%. On Days 1, 5, and 7 we determined glucose and lactate concentration, pH, P-selectin expression, hypotonic shock response (HSR), and extent of shape change (ESC). Clotting Factor V (FV) and VIII (FVIII) activities and D-dimer concentration were determined in CP and donor. RESULTS: Glucose utilization, lactate production, and pH were similar for both kinds of products. Low P-selectin expression indicated no relevant PLT activation during storage. HSR and ESC were similarly well preserved. Recoveries of FV and FVIII were 100.0±14.0 and 98.6±14.9%, respectively. Concentrations of D-dimers in the donor and CP were 173.7±90.1 and 177.6±91.2 ng/dL, respectively. CONCLUSIONS: Adding PAS immediately or 2 hours after collection does not result in different in vitro quality of PLTs stored up to 7 days. The good recovery of clotting factors with no signs of activation indicates a good quality of CP.

High resolution multimer analysis and the PFA-100 platelet function analyser can detect von Willebrand disease type 2A without a pathological ratio of ristocetin cofactor activity and von Willebrand antigen level.

BACKGROUND: The biological variability of von Willebrand factor and the variability in assays can make diagnosis and subclassification of von Willebrand disease (VWD) difficult. We describe a case series of four patients with a typical history of VWD and prolonged closure time in the platelet function analyser (PFA-100) but initially a normal ratio of ristocetin cofactor activity (VWF:RCo) to von Willebrand factor antigen levels (VWF:Ag) for whom further diagnostics verified VWD type 2A. METHODS: For the initial VWD diagnostics we measured VWF:Ag, VWF:RCo, platelet aggregation induced by ADP, ristocetin and collagen, closure time in the PFA-100 test, and platelet count. We used VWF multimer analysis and collagen binding capacity for extended diagnostics. VWD diagnostics were carried out as part of extensive laboratory screening to exclude other haemostatic defects. RESULTS: Multimer analysis revealed the absence of ultralarge multimers in all 4 patients. Ristocetin-induced platelet aggregation was consistently diminished in three patients with hereditary VWD 2A but not in a patient with essential thrombocythaemia. After repeat testing, diminished VWF:RCo and collagen binding capacity (VWF:CB) could be identified in all patients. However, all four cases would have been missed if the initial VWD assays had been performed only once. CONCLUSIONS: A single measurement of a normal ratio of VWF:RCo/VWF:Ag does not exclude VWD 2A in patients with a typical history of VWD. The PFA-100 is suitable for screening. To ensure that no cases of VWD are missed, multimer analysis and repeat functional testing of platelet aggregation, VWF:RCo, and VWF:CB are necessary.

Centrifugation after irradiation of red blood cells does not accelerate haemolysis.

BACKGROUND: For intrauterine transfusion and some other rare indications, irradiation and washing or adjustment to an elevated haematocrit is necessary. No data are currently available indicating whether irradiation of red blood cell concentrates (RBCs) might impair the mechanical stability of erythrocytes during centrifugation leading to elevated haemolysis. Consequently, if irradiation and centrifugation of RBCs is necessary, there is no definitive recommendation about the preferred sequence of steps. METHODS: We divided 20 RBC units that were not older than 9 days into two subunits. These subunits were prepared to yield irradiated RBCs with an elevated haematocrit, as they are used for intrauterine transfusion. One subunit was centrifuged and then irradiated, the other subunit was irradiated and then centrifuged. The units were evaluated in vitro before preparation and on days 1 and 7. RESULTS: We could not find any difference in the haemolysis rate, extracellular LDH or alpha-HBDH between the two groups of RBCs. This observation indicates that centrifugation after irradiation of RBCs does not accelerate haemolysis. A similar ATP content in the two subunits demonstrated no difference in energy metabolism. The extracellular potassium concentration was significantly lower in the subunits washed after irradiation. CONCLUSIONS: There is no difference in the haemolysis caused by centrifugation between irradiated and non-irradiated RBCs. However, it is well known that washing RBCs after irradiation significantly lowers the potassium content. Summarising these two findings leads to the conclusion that it is optimal first to irradiate and then to wash RBCs.

Thromboembolic risk assessment in a phenotypically cured factor-V-Leiden carrier after orthotopic liver transplantation.

BACKGROUND: The factor V Leiden mutation is a common genetic risk factor for thromboembolism. After liver transplantation, patients may present with an acquired factor V phenotype - genotype discrepancy. CASE REPORT: We present the history of a heterozygous carrier of the factor V Leiden mutation who needed liver transplantation because of coumarin-induced acute liver failure. This led to a phenotype - genotype discrepancy with apparent cure from the factor V Leiden carrier status. CONCLUSIONS: To date the thromboembolic risk assessment regarding the need for postoperative prophylactic anticoagulation in such patients has remained controversial with respect to the intracellular fraction of factor V in platelets. However, recent observations have shown that platelet factor V Leiden is endocytosed by megacaryocytes from plasma. Therefore, former assessments of an ongoing risk for thromboembolic events despite apparent cure of the factor V Leiden carrier status after liver transplantation should be corrected.