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.

Principles of Therapeutic Apheresis in Neurological Disease.

Background: Therapeutic plasma exchange (TPE) is a well-known apheresis technology since many years and is available worldwide. Myasthenia gravis is one of the first neurological diseases successfully treated with TPE. TPE is also frequently applied in acute inflammatory demyelinating polyradiculoneuropathy (Guillain-Barré syndrome). Both neurological disorders are immunologically mediated and might cause life-threatening symptoms in patients. Summary: There is a large body of evidence from many randomized controlled trials (RCTs) that the application of TPE in myasthenia gravis crisis or in acute Guillain-Barré syndrome is effective and safe. Thus, TPE is recommended as first-line therapy with a grade 1A recommendation during the critical course of these neurological diseases. Even chronic inflammatory demyelinating polyneuropathies characterized by complement-fixing autoantibodies to myelin are successfully treated with TPE. The plasma exchange reduces inflammatory cytokines, complements activating antibodies, and leads to an improvement of neurological symptoms. TPE is no standalone treatment but often combined with immunosuppressive therapy. Recent studies (clinical trials, retrospective analysis, meta-analysis, and systematic reviews) evaluate special apheresis technology (i.e., immunoadsorption [IA], small volume plasma exchange), compare different treatments of these neuropathies, or report on the therapy of rare immune-mediated neuropathies in case reports. Key Messages: TA is a well-established treatment and is safe in acute progressive neuropathies (myasthenia gravis, Guillain-Barré syndrome) with an immune etiology. TPE has been applied for decades and thus has the best evidence so far. The indication for IA depends on the availability of that technology and the evidence by RCTs in special neurological diseases. The treatment with TA should improve the clinical outcome of patients, reducing acute or chronic (chronic inflammatory demyelinating polyneuropathies) neurological symptoms. The informed consent of the patient should carefully weight risks and benefits of the apheresis treatment and consider alternative therapies.

Head and neck tumor cells treated with hypofractionated irradiation die via apoptosis and are better taken up by M1-like macrophages.

PURPOSE: The incidence of head and neck squamous cell carcinomas (HNSCC) is increasing worldwide, especially when triggered by the human papilloma virus (HPV). Radiotherapy has immune-modulatory properties, but the role of macrophages present in HNSCC and having contact with irradiated tumor cells remains unclear. The influence of irradiated (2â€¯× 5Gy) HNSCC cells on the (re-)polarization and phagocytosis of human macrophages, either non-polarized or with a more M1 or M2 phenotype, was therefore investigated. METHODS: Human monocytes were differentiated with the hematopoietic growth factors M­CSF (m) or GM-CSF (g) and additionally pre-polarized with either interleukin (IL)-4 and IL-10 or interferon (IFN)-γ and lipopolysaccharides (LPS), respectively. Subsequently, they were added to previously irradiated (2â€¯× 5Gy) and mock-treated HPV-positive (UD-SCC-2) and HPV-negative (Cal33) HNSCC cells including their supernatants. RESULTS: The HNSCC cells treated with hypofractionated irradiation died via apoptosis and were strongly phagocytosed by M0m and M2 macrophages. M0g and M1 macrophages phagocytosed the tumor cells to a lesser extent. Irradiated HNSCC cells were better phagocytosed by M1 macrophages compared to mock-treated controls. The polarization status of the macrophages was not significantly changed, except for the expression of CD206 on M2 macrophages, which was reduced after phagocytosis of irradiated HPV-negative cells. Further, a significant increase in the uptake of irradiated HPV-positive cells by M0g macrophages when compared to HPV-negative cells was observed. CONCLUSION: HNSCC cells treated with hypofractionated irradiation foster phagocytosis by anti-tumorigenic M1 macrophages. The data provide the first evidence on the impact of the HPV status of HNSCC cells on the modulation of the macrophage response to irradiated tumor cells.

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.

Blood counts in adult and elderly individuals: defining the norms over eight decades of life.

The blood count is one of the most common tests used for health assessment. In elderly individuals, selection of a 'healthy' reference population for laboratory assessment is difficult due to the high prevalence of chronic morbidities, leading to uncertainty regarding appropriate reference intervals. In particular, age-specific lower haemoglobin reference limits to define anaemia are controversial. Here, we applied a data mining approach to a large dataset of 3 029 904 clinical routine samples to establish blood count reference intervals. We excluded samples from units/specialists with a high proportion of abnormal blood counts, samples from patients with an unknown or decreased estimated glomerular filtration rate, and samples with abnormal test results in selected other analytes. After sample exclusion, 566 775-572 060 samples from different individuals aged 20-100 years were available for analysis. We then used an established statistical algorithm to determine the distribution of physiological test results and calculated age- and sex-specific reference intervals. Our results show substantial trends with age in haematology analytes' reference intervals. Most notably, haemoglobin and red cell counts decline in men with advanced age, accompanied by increases in red cell volume in both sexes. These findings were confirmed in an independent dataset, and suggest an at least partly physiologic cause.

Thrombocyte apheresis cassettes as a novel source of viable peripheral blood mononuclear cells.

BACKGROUND: Traditionally, white blood cells (WBCs) are collected from buffy coats or freshly drawn blood. However, the increasing demand for peripheral blood mononuclear cells (PBMCs) in the research phases of immunological therapy development makes it necessary to identify alternative sources of these cells. STUDY DESIGN AND METHODS: Leukapheresis products are cost intensive and not offered by all blood banks. Therefore, thrombocyte apheresis cassettes (TACs), plateletpheresis waste products, were investigated as a possible low-cost and easily accessible blood source for research laboratories. The recovery rate, phenotype, and functionality of WBC subsets from TAC are unknown and were investigated in comparison to frequently used blood resources via flow cytometry. RESULTS: On average, TACs provide 30.3 × 106 /mL PBMCs, situating themselves between peripheral whole blood (WB; 5.35 × 106 /mL) and leukoreduction system chamber (LRSC; 163.9 × 106 /mL) yields. Frequencies of CD14, CD3, CD4, CD8, CD56, CD19, and CD11c positive cells in TACs correlate with normal proportions of WBC populations. Stimulation of TAC-derived PBMCs by lipopolysaccharide (LPS) and resiquimod (R848) showed no significant differences in expression levels of human leukocyte antigen (HLA)-DR, DQ, DP, and CD86 or cytokine secretion compared to other blood source derived PBMC. Following stimulation with LPS or R848, comparable levels of tumor necrosis factor-α, interleukin-10, and interleukin-1ß could be measured between TAC, LRSC, and WB. Additionally, TAC-derived T cells retained their proliferation capability and were able to produce interferon-γ following T-cell receptor stimulation. CONCLUSION: TACs provide a cost-effective source of viable and functional human blood cells that can readily be used for clinical and laboratory investigations after plateletpheresis preparation.

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.

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.

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.

Recommendations for Therapeutic Apheresis by the Section "Preparative and Therapeutic Hemapheresis" of the German Society for Transfusion Medicine and Immunohematology.

The section "Preparative and Therapeutic Hemapheresis" of the German Society for Transfusion Medicine and Immunohematology (DGTI) has reviewed the actual literature and updated techniques and indications for evidence-based use of therapeutic apheresis in human disease. The recommendations are mostly in line with the "Guidelines on the Use of Therapeutic Apheresis in Clinical Practice" published by the Writing Committee of the American Society for Apheresis (ASFA) and have been conducted by experts from the DACH (Germany, Austria, Switzerland) region.

Impact of collection programs for the generation of monocyte apheresis products on product quality and composition as starting material for the generation of cellular therapeutics.

BACKGROUND: With the discontinuation of the last generation of apheresis machines, new options for monocyte apheresis became available. As apheresis products play a crucial role in the generation of new cellular therapeutics (e.g., generation of dendritic cells [DCs] or precursor for T-cell experiments) we sought to compare two different collection programs for potential benefits or disadvantages due to different composition of the cellular products. STUDY DESIGN AND METHODS: Composition of discontinuously and continuously collected monocyte products from the same 13 donors was analyzed. For further evaluation as starting material for manufacturing of cellular therapeutics typically used steps such as Ficoll Isopaque, cryoconservation and monocyte isolation, with subsequent generation of mature DCs (mDCs) and assessment of T-cell function, were performed on seven of these apheresis pairs. RESULTS: Yield of total cells, monocytes, and mDCs was equal with both methods. T-cell composition did not differ significantly in content of CD3+, CD4+, and CD8+ cells. Differentiation status and cytokine production of CD8+ T cells upon stimulation with cytomegalovirus pp65 antigen was not significantly different. CONCLUSION: Both methods seem comparably suited for the generation of cellular products. If the intended use is "fresh" (without cryoconservation), continuously harvested cells show better cell numbers, while discontinuously harvested cells show better recovery after cryoconservation.

Contribution of MINCLE-SYK Signaling to Activation of Primary Human APCs by Mycobacterial Cord Factor and the Novel Adjuvant TDB.

Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, is an abundant cell wall glycolipid and major virulence factor of Mycobacterium tuberculosis. Its synthetic analog trehalose-6,6-dibehenate (TDB) is a new adjuvant currently in phase I clinical trials. In rodents, the C-type lectin receptors Mincle and Mcl bind TDB/TDM and activate macrophages and dendritic cells (DC) through the Syk-Card9 pathway. However, it is unknown whether these glycolipids activate human innate immune cells through the same mechanism. We performed in vitro analysis of TDB/TDM-stimulated primary human monocytes, macrophages, and DC; determined C-type lectin receptor expression; and tested the contribution of SYK, MINCLE, and MCL by small interfering RNA knockdown and genetic complementation. We observed a robust chemokine and cytokine release in response to TDB or TDM. MCSF-driven macrophages secreted higher levels of IL-8, IL-6, CCL3, CCL4, and CCL2 after stimulation with TDM, whereas DC responded more strongly to TDB and GM-CSF-driven macrophages were equally responsive to TDB and TDM. SYK kinase and the adaptor protein CARD9 were essential for glycolipid-induced IL-8 production. mRNA expression of MINCLE and MCL was high in monocytes and macrophages, with MINCLE and MCL proteins localized intracellularly under resting conditions. Small interfering RNA-mediated MINCLE or MCL knockdown caused on average reduced TDB- or TDM-induced IL-8 production. Conversely, retroviral expression in murine Mincle-deficient DC revealed that human MINCLE, but not MCL, was sufficient to confer responsiveness to TDB/TDM. Our study demonstrates that SYK-CARD9 signaling plays a key role in TDB/TDM-induced activation of innate immune cells in man as in mouse, likely by engagement of MINCLE.

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.

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.

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.

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 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.

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.