Validation of a Parvovirus B19 NAT Assay for Screening of Umbilical Cord Blood for Allogenic Hematopoietic Stem Cell Donation.

Introduction: Parvovirus B19 transmitted by umbilical cord blood (UCB) products may cause severe disease in allogenic hematopoietic stem cell transplant recipients. Thus, commercially available nucleic acid test (NAT) assays for highly sensitive detection of parvovirus B19 DNA validated for the specimen cord blood plasma (CBP) are required to avoid parvovirus B19 transmission by umbilical hematopoietic stem cell preparations. Methods: The multiplex cobas DPX NAT assay was validated for detection of parvovirus B19 DNA in CBP derived from citrate anticoagulated UCB units which have been processed by the Rubinstein method. In total, 363 retained CBP samples pretested negative for parvovirus B19 DNA were prepared for analyzing sensitivity, specificity, and interference of that NAT assay. The 3rd WHO International Standard for parvovirus B19 DNA was used for determining the 95% limit of detection (LOD95) by probit analysis. Results: The validation of the parvovirus B19 NAT assay for CBP demonstrated high sensitivity, specificity, intra- and inter-assay precision. Dilution series and replicate analyses showed a high linearity of the assay with a coefficient of determination above 0.99 and revealed a LOD95 of 17 International Units (IU)/mL (95% confidence interval, 14-44 IU/mL) for parvovirus B19 DNA in CBP samples. Conclusion: The validation of a commercially available parvovirus B19 NAT assay for the specimen CBP demonstrated a high assay performance fulfilling German guidelines and international regulations.

Validation of a SARS-CoV-2 RNA RT-PCR assay for high-throughput testing in blood of COVID-19 convalescent plasma donors and patients.

BACKGROUND: The frequency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNAemia in blood donors is uncertain. Thus, assays for SARS-CoV-2 RNA detection in blood, validated on commercially available polymerase chain reaction (PCR) systems, are required to allow a good comparability of data. STUDY DESIGN AND METHODS: The cobas SARS-CoV-2 dual-target reverse transcriptase PCR (RT-PCR) assay, licensed for respiratory swab SARS-CoV-2 RNA testing, was validated for detection of viral RNA in blood. For the validation panel, SARS-CoV-2-positive plasma samples were prepared by spiking SARS-CoV-2-positive respiratory specimens in negative human plasma. The 95% limit of detection (LOD95) was determined by probit analysis. For clinical validation, coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) donors and patients with COVID-19 with a severe disease course treated in an intensive care unit (ICU) were included. RESULTS: The validation of the SARS-CoV-2 RT-PCR assay for blood demonstrated high sensitivity and specificity and intra- and inter-assay precision and efficiency. The LOD95 for SARS-CoV-2 RNA was 5.0 genome copies/mL (95% confidence interval [CI], 3.3-12 copies/mL) for target 1 and 4.3 genome copies/mL (95% CI, 2.9-10 copies/mL) for target 2. In a cohort of 39 CCP donors with 66 CCP donations no SARS-CoV-2 RNA in plasma was detected. Screening of 25 blood samples of 19 ICU patients with COVID-19 showed six positive results for SARS-CoV-2 RNA in at least one target of the assay. CONCLUSION: The SARS-CoV-2 RNA assay, only licensed for respiratory swabs, performed on a PCR system for high-throughput testing, showed a good assay performance for blood testing.

Thrombin Generation in Fresh and Frozen-Thawed Platelet Poor Plasma - Is there a Difference?

BACKGROUND: Applicability of thrombin generation tests to clinical routine has been sought for many years. The aim of this study was to compare thrombin generation measured in fresh platelet poor plasma (f-PPP) and frozen-thawed platelet poor plasma (ft-PPP) to prove the consistency of results. METHODS: In this prospective study, thrombin generation was measured in twenty-fold repetitions in 3.2% citrate PPP obtained from male healthy blood donors aged 19 - 39 years (n = 54 donations). The tests were performed with fresh PPP and repeated after storing the PPP at -60°C. In two subgroup analyses, the effect of higher and lower normal baseline platelet counts on the Calibrated Automated Thrombogram (CAT) assay and the influence of ABO blood groups on thrombin generation were analyzed. RESULTS: Referring to the parameters of thrombin generation most frequently used in studies, peak thrombin of f-PPP and ft-PPP agreed in about 50% of the samples. Endogenous thrombin potential (ETP) of f-PPP and ft-PPP agreed in nearly two-thirds of the samples. A slightly but significantly slower kinetic was found in the thrombin generation of ft-PPP compared with f-PPP. At least in f-PPP, ETP correlates with baseline platelet counts of the whole blood sample. Peak thrombin was significantly higher in non-O blood groups compared to O blood group. CONCLUSIONS: A low level of agreement between the results of f-PPP and ft-PPP is shown. In terms of practicability of sample collection using semi-automated thrombin-generation assays ft-PPP should be preferred over f-PPP. We therefore recommend using ft-PPP in clinical studies.

Ischemic stroke and dose adjustment of oral Factor Xa inhibitors in patients with atrial fibrillation.

BACKGROUND: Oral Factor Xa inhibitors for the prevention of stroke in atrial fibrillation require dose adjustment based on certain clinical criteria, but the off-label use of the reduced doses is common. METHODS: Data from an observational registry including patients admitted with acute cerebral ischemia while taking oral Factor Xa inhibitors for atrial fibrillation between April 2016 and December 2018 were investigated. The dose regimen of the Xa inhibitor was classified as "appropriate", "underdosed" and "overdosed" in conformity with the European Medicines Agency labelling. The effect of underdosing on the functional factor Xa plasma level on admission, the clinical stroke severity and the functional outcome after 3 months were investigated. RESULTS: 254 patients with cerebral ischemia while on Factor Xa inhibitors were included. The dose regimen of the Factor Xa inhibitor was appropriate in 166 patients (65%), underdosed in 67 patients (26%) and overdosed in 21 patients (8%). Underdosing was associated with female sex, diabetes mellitus and higher CHA2DS2-Vasc scores. Underdosing independently predicted lower anti-Xa plasma levels on admission [median 69.4 ng/ml (IQR 0.0-121.6) vs. 129.2 ng/ml (65.5-207.2); p < 0.001], was associated with higher NIHSS scores on admission [median 5 (IQR 1-10) vs. 3 (1-7); p = 0.041] and worse functional outcome after 3 months (favorable outcome 26.9% vs. 46.9%; p = 0.025). CONCLUSION: One in three patients with ischemic stroke during treatment with oral Xa inhibitors used inappropriate dose regimens. Underdosing was associated with lower functional plasma levels, higher clinical stroke severity and worse functional outcome.

Direct Oral Anticoagulant Plasma Levels for the Management of Acute Ischemic Stroke.

INTRODUCTION: The management of acute ischemic stroke in patients on direct oral anticoagulants (DOACs) is challenging. However, the substance-specific plasma level could guide treatment decisions on recanalization therapies. We present a plasma-level-based protocol for emergency treatment of stroke patients on oral anticoagulants. Bleeding complications and clinical outcome for patients on DOACs are reported and compared to patients on vitamin K antagonists (VKAs). METHODS: In patients with acute ischemic stroke and suspected use of DOACs within 48 h prior to hospital admission, plasma levels were measured using the calibrated Xa-activity (apixaban, edoxaban, rivaroxaban) or the Hemoclot®-assay (dabigatran). Levels <50 ng/mL were supportive for thrombolysis, while high values >100 ng/mL excluded patients from recombinant tissue plasminogen activator use. For patients on VKAs, the cutoff was set at international normalized ratio of 1.7. Endovascular thrombectomy of a large vessel occlusion was performed independently from coagulation testing. Consecutive patients were included in an observational registry. RESULTS: Five hundred and twenty-two patients (261 on VKAs and 261 on DOACs) were included. Thirty patients (11.5%) on VKAs and 24 (9.2%) on DOACs received thrombolysis, followed by mechanical thrombectomy in 10 and 14 patients, respectively. Seventeen patients in each group received thrombectomy only. Symptomatic intracranial hemorrhage associated with thrombolysis occurred in 1 patient on VKA (3.3%) and 1 on DOAC (4.2%; p = 0.872). The turnaround time of specific assays did not show a significant delay in comparison to standard coagulation parameters. CONCLUSION: DOAC plasma levels could support decisions on emergency treatment of ischemic stroke. Systemic thrombolysis below suggested thresholds appears preliminary feasible and safe without an excess in bleeding complications.

Multivariate Platelet Analysis Differentiates Between Patients with Alzheimer's Disease and Healthy Controls at First Clinical Diagnosis.

BACKGROUND: Early diagnosis of Alzheimer's disease (AD) is challenging, and easily accessible biomarkers are an unmet need. Blood platelets frequently serve as peripheral model for studying AD pathogenesis and might represent a reasonable biomarker source. OBJECTIVE: In the present study, we investigated the potential to differentiate AD patients from healthy controls (HC) based on blood count, platelet morphology, and function as well as molecular markers at the time of first clinical diagnosis. METHODS: Blood samples from 40 AD patients and 29 age-matched HC were included for determination of 78 parameter by blood counting, platelet morphometry, aggregometry, flow cytometry (CD62P, CD63, activated fibrinogen receptor), protein quantification of nicotinic acetylcholine receptor α7 (nAChRα7) and caveolin-1 (CAV-1), and miRNA quantification (miR-26b, miR-199a, miR-335). Group comparison between patients and controls was performed in univariate and multivariate statistical analyses. RESULTS: AD patients showed significantly lower aggregation response to ADP and arachidonic acid and significantly decreased CD62P and CD63 surface expression induced by ADP and U46619 compared to HC. Relative nAChRα7 and CAV-1 expression was significantly higher AD platelets than in HC. Multivariate analysis of 63 parameter revealed significant differences between AD patients and healthy controls. The best performing feature model revealed a sensitivity of 96.6%, a specificity of 80.0%, and a positive predictive value of 89.3%. No grouping could be achieved by using single parameter groups. CONCLUSION: Significant differences between platelet characteristics from AD patients and HC at the time of first clinical diagnosis were observed. The best performing parameter can be used as a blood-based biomarker for AD diagnosis in a multivariate model in addition to the standardized mental tests.

Cerebral Ischemia in Patients on Direct Oral Anticoagulants.

Background and Purpose- In patients with ischemic stroke on therapy with vitamin K antagonists, stroke severity and clinical course are affected by the quality of anticoagulation at the time of stroke onset, but clinical data for patients using direct oral anticoagulants (DOACs) are limited. Methods- Data from our registry including all patients admitted with acute cerebral ischemia while taking oral anticoagulants for atrial fibrillation between November 2014 and October 2017 were investigated. The activity of vitamin K antagonists was assessed using the international normalized ratio on admission and categorized according to a threshold of 1.7. DOAC plasma levels were measured using the calibrated Xa-activity (apixaban, rivaroxaban, and edoxaban) or the Hemoclot-assay (dabigatran) and categorized into low (<50 ng/mL), intermediate (50-100 ng/mL), or high (>100 ng/mL). Primary objective was the association between anticoagulant activity and clinical and imaging characteristics. Results- Four hundred sixty patients were included (49% on vitamin K antagonists and 51% on DOAC). Patients on vitamin K antagonists with low international normalized ratio values had higher scores on the National Institutes of Health Stroke Scale and a higher risk of large vessel occlusion on admission. For patients on DOAC, plasma levels were available in 75.6% and found to be low in 49 (27.7%), intermediate in 41 (23.2%), and high in 87 patients (49.2%). Low plasma levels were associated with higher National Institutes of Health Stroke Scale scores on admission (low: 8 [interquartile range, 3-15] versus intermediate: 4 [1-11] versus high: 3 [0-8]; P<0.001) and higher risk of persisting neurological deficits or cerebral infarction on imaging (85.7% versus 75.6% versus 54.0%; P<0.001). Low DOAC plasma levels were an independent predictor of large vessel occlusion (odds ratio, 3.84 [95% CI, 1.80-8.20]; P=0.001). Conclusions- The activity of anticoagulation measured by specific DOAC plasma levels on admission is associated with stroke severity and presence of large vessel occlusion.

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.

Comparative morphology analysis of live blood platelets using scanning ion conductance and robotic dark-field microscopy.

Many conventional microscopy techniques for investigating platelet morphology such as electron or fluorescence microscopy require highly invasive treatment of the platelets such as fixation, drying and metal coating or staining. Here, we present two unique but entirely different microscopy techniques for direct morphology analysis of live, unstained platelets: scanning ion conductance microscopy (SICM) and robotic dark-field microscopy (RDM). We demonstrate that both techniques allow for a quantitative evaluation of the morphological features of live adherent platelets. We show that their morphology can be quantified by both techniques using the same geometric parameters and therefore can be directly compared. By imaging the same identical platelets subsequently with SICM and RDM, we found that area, perimeter and circularity of the platelets are directly correlated between SICM and dark-field microscopy (DM), while the fractal dimension (FD) differed between the two microscopy techniques. We show that SICM and RDM are both valuable tools for the ex vivo investigation of the morphology of live platelets, which might contribute to new insights into the physiological and pathophysiological role of platelet spreading.

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.

The influence of pre-analytical conditions on platelet-derived microparticles.

BACKGROUND: Microparticles (MP) have recently become a focus of both research and clinical investigations. As pre-analytical conditions frequently remain unpublished, further studies are needed to analyze their impact on MP release. METHODS: This prospective study investigated the effect of sequential storage under three different sets of conditions (fresh; storage at 4 degrees C for 24 hours, SC1; storage at -70 degrees C for 24 hours, SC2) and agitation on platelet-derived MP (PMP) in 11 healthy blood donors (6 male, 5 female). PMP were quantified using flow cytometry (FCM) for analysis of all events positive for both CD41a-PE and Annexin-V-FITC. Newly developed calibration beads for FCM (size of 0.3 - 0.9 microm) were applied for FCM. For functional testing a phospholipid-dependent clotting assay (XACT) was used. RESULTS: PMP concentration increased 1.7-fold in platelet-poor plasma (PPP) under SC1 and further increased 1.6-fold (p < 0.001) under SC2 (p = 0.005). Overall, samples of SC2 had a 5.5-fold increased count of large PMP (0.5 - 0.9 microm) compared to baseline. Results in samples of SC2 ranged from 40.1 seconds to 80.3 seconds but on average the CT was also shortened compared to the CT for SC1 and fresh samples. Additional agitation before PPP preparation reduced the PMP concentration by around 50% (p = 0.025). 135% more small PMP were detected with recently developed calibration beads. Compared to CT (XACT) flow cytometry using Megamix Plus calibration beads is able to reveal significant differences between the analyzed preanalytical conditions. CONCLUSIONS: Fresh blood samples should be used for standardizing PMP analysis. Calibration beads for FCM (size of 0.3 - 0.9 microm) have shown to be a reliable tool for PMP quantification especially for PMP of smaller sizes up to 300 nm. Agitation of blood samples before PMP analysis should be avoided. The application of XACT is limited for the analysis of preanalytical conditions.

Fractal and Euclidean descriptors of platelet shape.

Platelet shape change is a dynamic membrane surface process that exhibits remarkable morphological heterogeneity. Once the outline of an irregular shape is identified and segmented from a digital image, several mathematical descriptors can be applied to numerical characterize the irregularity of the shapes surface. 13072 platelet outlines (PLO) were segmented automatically from 1928 microscopic images using a newly developed algorithm for the software product Matlab R2012b. The fractal dimension (FD), circularity, eccentricity, area and perimeter of each PLO were determined. 972 PLO were randomly assigned for computer-assisted manual measurement of platelet diameter as well as number, width and length of filopodia per platelet. FD can be used as a surrogate parameter for determining the roughness of the PLO and circularity can be used as a surrogate to estimate the number and length of filopodia. The relationship between FD and perimeter of the PLO reveals the existence of distinct groups of platelets with significant structural differences which may be caused by platelet activation. This new method allows for the standardized continuous numerical classification of platelet shape and its dynamic change, which is useful for the analysis of altered platelet activity (e.g. inflammatory diseases, contact activation, drug testing).

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.

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.

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.

A New Method for Measuring the Dynamic Shape Change of Platelets.

BACKGROUND: Platelet shape change is a dynamic process that has been classified in different types. Exact documentation of platelet structure needs an improved method of measuring platelet shape. METHODS: 10 µl of platelet-rich plasma (PRP) from anticoagulated whole blood (3.2% buffered sodium citrate 0.105 mol/l) was put onto a glass slide covered with a cover slip. By use a of dark field light microscope connected with a CMOS-Camera a photographic snap-shot was taken after 5 and 30 min. Diameter of platelets and length of filopodia were measured with a self-developed plugin for ImageJ software. Statistic calculation was performed with Excel WinSTAT Microsoft software. RESULTS: We showed a swelling of the granulomer from 2.06 ± 0.56 µm to 2.33 ± 0.59 µm (p < 0.05), a reduction of pseudopodia (2.10 ± 0.94 vs. 1.78 ± 1.04 µm; p < 0.05) in conjunction with an increase of hyalomer diameter from 3.29 ± 0.83 to 3.50 ± 0.85 µm (p < 0.05), and an increase of pseudopodia length from 2.68 ± 1.45 µm to 3.67 ± 1.79 µm (p < 0.005) in conjunction with an increase of hyalomer diameter from 6.58 ± 1.91 µm to 7.94 ± 1.87 µm (p < 0.05). CONCLUSION: We revealed and documented a dynamic change of platelet size and filopodia structure in PRP. This method allows an exact analysis of platelet size and surface structures.