Standard Platelet Research Articles

Successful desensitization to immunoglobulin A in a case of transfusion‐related anaphylaxis

A history of anaphylaxis after transfusion of immunoglobulin A (IgA)-containing blood products in selective IgA-deficient (sIgAD) patients can be a major problem, particularly in emergencies, when large quantities of blood products are required. A 19-year-old woman with end-stage Type 2 autoimmune hepatitis required liver transplantation as her only remaining treatment option. However, she also had sIgAD, anti-IgA antibodies, and episodes of anaphylaxis after receiving IgA-containing blood products. Liver transplantation would have been extremely challenging due to the difficulty of obtaining sufficient blood products from suitable IgA-deficient donors. Hence, it became imperative to devise a protocol to desensitize her to IgA-containing blood products. Using a continuous infusion of an IgA-enriched (6 mg/mL IgA) immunoglobulin preparation with gradual increases in concentration, she was successfully desensitized to IgA. Consequently, she was able to receive standard platelets, fresh-frozen plasma, and red blood cells with no complications. This approach could prove very useful in similar cases that may require administration of large quantities of blood products particularly in emergency lifesaving circumstances.

Platelet apoptosis and activation in platelet concentrates stored for up to 12 days in plasma or additive solution

Several studies suggest that apoptosis of platelets occurs during storage of platelet concentrates (PC). We sought to determine whether storage of PC in additive solution alters levels of apoptosis during storage beyond the current shelf life (5-7 days). Pooled buffy coat PC (n = 7) were prepared in either 100% plasma or 70% Composol and stored at 22 °C for 12 days. A third arm of the study stored PC in 100% plasma at 37 °C, which is thought to induce apoptosis. PC were tested for mitochrondrial membrane potential, annexin V binding, microparticles, caspase-3/7 activity and decoy cell death receptor 2, as well as standard platelet quality tests. Composol units remained ≥pH 6·88, with 36% lower lactate and higher pH vs plasma by day 12 (P < 0·001). Platelet function was better maintained, and activation and apoptotic markers tended to be lower in Composol units towards the end of storage. However, levels of all apoptosis markers assessed were not significantly different in units stored in Composol. Storage at 37 °C saw stronger correlation of apoptotic markers with standard quality tests compared to 22 °C, but loss of correlation of caspase-3/7 activity with other apoptosis markers. We conclude that storage of platelets in 70% Composol vs 100% plasma does not increase the rate of platelet apoptosis. Our data agree with other studies suggesting that platelet apoptosis is sequential to high levels of activation, but share a significant degree of overlap.

Thrombospondin-1: a unique marker to identify in vitro platelet activation when monitoring in vivo processes.

Measuring platelet activation in patients has become a potent method to investigate pathophysiological processes. However, the commonly applied markers are sensitive to detrimental influences by in vitro platelet activation during blood analysis. Protein isoforms of platelet-derived thrombospondin-1 (TSP-1) were investigated for their potential to identify in vitro platelet activation when monitoring in vivo processes. TSP-1 was determined in plasma, serum or supernatant of purified platelets by ELISA and immunoblotting and was compared with standard markers of platelet activation. A collective of 20 healthy individuals and 30 cancer patients was analyzed. While in vitro platelet degranulation led to a selective increase in the 200-kDa full-length molecule, an in vivo process involving platelet activation such as wound healing resulted in the predominant rise of the 140-kDa TSP-1 protein. The physiological ratio of circulating TSP-1 variants was determined and a cut-off level at 1.0 was defined to identify plasma samples with artificial in vitro platelet activation exceeding the cut-off level. In contrast, cancer patients known to frequently exhibit increased in vivo activation of platelets presented with a significantly decreased ratio of TSP-1 variants as compared with healthy volunteers. In comparison to standard platelet markers, TSP-1 constitutes a sensitive and stable parameter suited to monitor in vitro platelet activation. The analysis of TSP-1 protein isoforms further offers a valuable tool to reliably discriminate between in vitro and in vivo effects, to exclude variability introduced during blood processing and improve clinical monitoring.

Analyse d’un incident bactérien grave transmis par transfusion d’un concentré plaquettaire

We recently observed a near fatal case of transfusion-transmitted infection with standard platelet concentrate. Streptococcus dysgalactiae subspecies equisimilis was isolated both from donor, residual component container and cultures of the patient's blood. This should question the usefulness of systematic bacterial detection in platelet concentrates, however a lethal accident has occurred recently which escaped bacterial detection. This observation calls for implementation of pathogen inactivation procedures for platelets concentrates.

In Vitro Megakaryocyte Production and Platelet Biogenesis: State of the Art

The exciting and extraordinary capabilities of stem cells to proliferate and differentiate into numerous cell types not only offers promises for changing how diseases are treated but may also impact how transfusion medicine may be practiced in the future. The possibility of growing platelets in the laboratory to some day supplement and/or replace standard platelet products has clear advantages for blood centers and patients. Because of the high utilization of platelets by patients undergoing chemotherapy or receiving stem cell transplants, platelet transfusions have steadily increased over the past decades. This trend is likely to continue as the number of adult and pediatric patients receiving stem cell transplants is also continuously rising. As a result of increased demand, coupled with the short shelf-life of platelet concentrates, providing platelets to patients can stretch the resources of most blood centers and drive donor recruitment efforts, and on occasion, platelet shortages can compromise the care of thrombocytopenic patients.

Menorrhagia in Adolescents with Platelet Function Disorders: Family History Suggests Further Investigations

Background: Platelet function disorders (PFDs) are a heterogeneous group of hemostatic defects that have received increasing attention as an etiology of adolescent menorrhagia. Diagnosing these disorders is complex and the laboratory testing is not well standardized. The evaluation often begins by examining the blood smear as well as assessing platelet counts and size. Standard platelet function screening tests are used. Platelet aggregation studies test for platelet function and are commonly used for confirming screening findings.

Utility of 96-well plate aggregometry and measurement of thrombi adhesion to determine aspirin and clopidogrel effectiveness

Aspirin and clopidogrel are key anti-thrombotic therapies. Results from platelet reactivity testing during therapy, have been shown to correlate with future events and would allow for the optimisation of therapy. However, there is little agreement among current tests and there remains a clear clinical need for a universal standardised test. It was the objective of this study to explore the potential of 96-well plate aggregometry as a definitive clinical test of platelet reactivity with respect to aspirin and clopidogrel. A small non-blinded trial of 16 healthy male volunteers assigned to seven days of aspirin (75mg/day) or clopidogrel (75mg/day) therapy. Blood was collected before and on day 7 of treatment. Platelet aggregation was measured using a 96-well plate based aggregation method, and thrombi adhesion measured by colourimetric assay. Platelet agonists used were ADP (0.1-30microM), arachidonic acid (0.03-1.3mM), collagen (0.1-30microg/ml), adrenaline (0.001-100microM), ristocetin (0.2-3mg/ml), TRAP6 amide (0.130microM) and U46619 (0.130microM). Concentration response curves were constructed to each agonist under the various conditions and used to extract data such as log EC(50), Hill slope, and area under the curve. These demonstrated low intra- and inter-assay variability and strong discrimination of drug effects. This study demonstrates the ability of the 96-well plate based aggregation and adhesion method to detect and differentiate between stable aspirin and clopidogrel treatment in healthy volunteers. Moreover, this assay marries the ability to test subjects or patients using a range of platelet agonists with more rapidity and ease than the current gold standard platelet assay, traditional light transmission aggregometry, making it a serious alternative assay for use in clinical settings.

Abstract 4017: Reduced Anti-Platelet Response to Clopidogrel in Diabetic Patients Undergoing Percutaneous Coronary Intervention

Despite dual antiplatelet therapy with aspirin and clopidogrel, patients with diabetes mellitus (DM) suffer from frequent recurrent ischemic events. Previous studies have shown that DM patients have a higher prevalence of aspirin resistance than non-DM patients. The aim of this analysis was to determine if DM patients have a decreased antiplatelet response to either maintenance or high loading clopidogrel administration when compared to non-DM patients. One hundred and thirty eight patients that underwent percutaneous coronary intervention (PCI) in the Clear Platelets-2 Study were included in this analysis. Patients were grouped according to clopidogrel dose use and presence of DM. Subjects were either on maintenance therapy with 75mg of clopidogrel (C75 group; n=72) or received a loading dose of 600mg of clopidogrel immediately after PCI (C600 group; n=66). All patients received 325-mg aspirin. Platelet function was measured by Light Transmission Aggregometry using ADP (5 and 20μM), TRAP (15 μM), and collagen (2μg/ml). Overall, DM patients in the C75 group had higher platelet aggregation using 5 and 20μM ADP and 2μg/ml collagen. DM patients had lower relative platelet inhibition at 24hrs with 5 μM ADP and 2μg/ml collagen in the C600 group when compared to non-DM patients (Table ). DM patients undergoing PCI exhibit higher platelet aggregation when receiving standard clopidogrel maintenance dose and lower relative platelet inhibition with high clopidogrel loading dose. Higher doses of clopidogrel or more potent P2Y12 receptor antagonists may be needed in DM patients to obtain comparable platelet inhibition to non-DM patients.

Characterization of the Pro-Thrombotic State in CAPD Patients

Purpose. To investigate whether a chronic pro-thrombotic tendency, which may contribute to a high rate of atherothrombotic disease, is present in patients treated for continuous peritoneal dialysis (CAPD), and, if so, what its pattern is. We investigated this issue by jointly exploring all the systems involved, the coagulation and fibrinolytic systems and platelets. Methods. Markers of coagulation activation, markers of fibrinolysis activation, and standard fibrinolytic parameters and platelet aggregation induced by different agents were measured in 15 patients treated by CAPD and in 15 matched, healthy controls. All CAPD patients received erythropoietin, were in the stable condition, and did not have acute disease or malignancy. Results. CAPD patients had substantially (p < 0.001) increased levels of prothrombin fragments F1+2, disclosing a low-grade activation of the coagulation system. D-dimer was also significantly (p < 0.05) increased, whereas the levels of t-PA antigen and activity, PAI antigen and activity, and plasminogen were comparable to controls, suggesting that slight secondary (and not primary) activation of fibrinolysis due to coagulation activation took place. Patients had significantly (p < 0.05) elevated levels of fibrinogen. A study of platelet aggregation (induced by adenosine diphosphate, collagen, and epinephrine) did not show platelet hyperactivity in patients. Conclusions. We found that a pro-thrombotic tendency is present in the plasma of CAPD patients. The main reason for a pro-thrombotic state is chronic low-grade activation of the coagulation system and elevated levels of fibrinogen. The fibrinolytic system and platelets seemingly do not contribute to this pro-thrombotic tendency.

Rab38 Expression in Platelet Dense Granule Storage Pool Disease.

Platelet dense granule storage pool disease (SPD) is a bleeding disorder characterized by a lack of normal platelet dense granule function, as evidenced by decreased platelet aggregation in response to ADP, epinephrine and collagen. Platelet SPD has been studied most extensively in humans and rodents with Hermansky-Pudlak syndrome (HPS), whose phenotype is a result of defects in granule trafficking, leading to oculocutanous albinism, lysosomal storage diseases, and platelet dysfunction. We have been characterizing the fawn-hooded hypertensive (FHH) rat, which has been previously shown to have a bleeding disorder consistent with a platelet SPD and some of the features of HPS. While the platelets in the FHH rat have normal alpha granules and lysosomes, they lack dense granules as assessed by transmission electron microscopy. Platelet flow cytometric analysis of GPIb and GPIIb indicated that the FHH platelets have normal surface expression of these adhesion proteins. The FHH rat has a mutation in the Rab38 gene at the ATG start site, which is associated with the bleeding disorder. Rab38 is part of a large family of GTPases, which are involved in granule formation and secretion. Western blotting of FHH tissues revealed that there is no expression of Rab38 protein. We have used confocal immunomicroscopy to assess Rab38 in platelet formation and function. In normal rat and human platelets, there was punctate expression of Rab38. There was no Rab38 staining detected in FHH platelets. In human megakaryocytic cell lines, Dami and HEL cells, there was punctate staining of Rab38 that was mainly in the periphery of the cells, with a variable amount of perinuclear staining. There was partial colocalization of Rab38 with serotonin and VWF, and with Lamp-3, a marker of lysosomes. The degree of colocalization varied between cells. There was no clear association of Rab38 with actin and tubulin in megakaryocytes. We also examined a cohort of patients with SPD, but not HPS, for mutations in Rab38. The entire coding region and intron-exon boundaries of the Rab38 gene were sequenced in 18 patient samples collected at Emory University for the CDC Women with Bleeding Disorders and Menorrhagia Study. Ten of the patients had platelet function defects documented by standard platelet aggregation studies, and eight had no identifiable platelet function defect. No mutations in Rab38 were detected. Whereas numerous known polymorphisms were identified and confirmed, there was no association of any of them with platelet function abnormalities. In conclusion, Rab38 is expressed in platelets and megakaryocytes and may interact with other granule proteins during megakaryocyte development. Failure to express Rab38 is associated with platelet dysfunction. Further studies are needed to determine its function in megakaryocytes and platelets, and to determine whether defects in Rab38 are a cause of platelet SPD in humans.

GPVI Mutation and Absent Platelet Collagen Activation in a Patient with a Bleeding Disorder.

The physiological relevance of the collagen GPVI receptor was known prior to its recognition as a platelet membrane receptor since several patients lacking GPVI as a consequence of autoantibody inhibition present with a mild bleeding diathesis. Remarkably, patients with a proven GPVI gene mutation could not yet been identified. We here describe a female patient with ecchymoses, epistaxis, several posttraumatic and postsurgery bleeding complications since her childhood and menorraghia. Coagulation and vWF studies were normal. The patient has a normal platelet count and electron microscopy showed no structural platelet abnormalities. All standard platelet aggregations were normal except for an absent response to Horm collagen, convulxin and the collagen-related peptide even at high concentrations of these agonists. Platelet ATP secretion was normal for ADP stimulation but absent for collagen. The mechanism of this dysfunction was further analyzed. Flow cytometric analysis of the different platelet membrane receptors showed a normal expression of integrin alphaIIb/beta3, integrin alpha2 and GPIbalpha but a completely absent membrane expression of GPVI. In contrast, immunoblot analysis of total platelet lysates showed a 50% reduced expression of both GPVI and its binding partner Fcgamma. Molecular analysis of the immunoreceptor GPVI/Fcgamma revealed no abnormalities in Fcgamma gene, except for the presence of Fcgamma mRNA with exon 2 skipping which could also be detected in control platelets. The patient was heterozygous for the known SKTQH/PEALN functional GPVI haplotype (Joutsi-Korhonen et al, Blood, 2003) but importantly also carried a heterozygous out-of-frame 16 bp deletion in exon 3 of GPVI gene. Subcloning and sequencing of the platelet GPVI mRNA resulted in only 25% of clones without the exon 3 deletion. The normal GPVI allele carried in addition to the SKTQH haplotype also 3 SNPs in the 3′UTR of the GPVI gene. The GPVI downstream intracellular signaling and collagen adhesion studies for this patient are ongoing. This study presents the first patient with a genetic GPVI defect with in contrast to patients with an autoantibody against GPVI a normal platelet count and ultrastructural morphology.

Normal Response to Aspirin in Healthy Individuals: Cross-Comparison of Light Transmission Aggregometry, VerifyNow™ System, Platelet Count Drop, Thrombelastography (TEG®) and Urinary 11-dehydrothromboxane B2.

Aspirin resistance is often defined as failure to demonstrate an inhibitory effect of aspirin on standard platelet function assays. Although many studies have been published on this phenomenon, the performance of individual assays to detect actual aspirin effects is poorly defined. The aim of this study is to define the sensitivity and potential limitations of five different assays of platelet function on detecting aspirin effects. A prospective cohort of 45 healthy volunteers consented to taking 80 mg of non-coated aspirin for 8–10 days. Participants were selected if they were non-smokers, did not present any significant medical condition and were excluded if they had ingested any potential drug / supplement known to effect platelet function in the past 10 days or were found to have abnormal blood counts. Samples were collected immediately prior to the first aspirin dose and repeated after 8 to 10 days of aspirin therapy. The study population consisted of 16 males and 29 females, 19 to 59 years of age without acquired cardiovascular risk factors and a mean BMI of 24,1 (range: 18,8–32,5). Study drug intake was carefully monitored by patient questionnaire and unused pill count at the second study visit. Partial non compliance was identified in 4 participants (1–2 missed doses) although no missed doses occurred in the last 3 days of the study. Mean delay between the last aspirin dose and the post study sample was 221 minutes (range 60 to 720 min, 5 patients with delays over 6 hours). Test characteristics and performances in detecting aspirin intake in our population are resumed in the table below.Conclusion: Although aspirin clearly has an effect in all the assays tested, the broad distribution of values with LTA-ADP, platelet count drop, TEG-MA(AA) and urinary dTxB2 does not allow accurate discrimination of aspirin effects in healthy individuals. Considering their adequate distinction between subjects on and off aspirin, we suggest that LTA-AA and the VerifyNow assay should be preferred in studies evaluating aspirin effect and potential resistance to this drug. Findings such as ours should serve to better define the limitations of laboratory studies of platelet function for the assessment of aspirin effects.Table 1Test performance characteristicsLTA-AA 1,6 mM (%)LTA-ADP 10 umol (%)VerifyNow (ARU)Platelet count drop (%)TEG-MAAA(%)dTxB2 (pg/ml)Prior to ASA61,2 (33,9;88,4)*74,2 (35,1;113,3)634,5 (564,8;704,3)74,9 (35,3;114,4)100,6 (27,2;174)240,8 (0;821)After ASA2,4 (0;7,9)40,4 (0;85,8)407,9 (355,7;460,1)41,5 (8,8;74,3)53,2 (−44,4;150,8)115 (0;407,9)p value**<10e-29<10e-13<10e-32<10e-10<10e-7<10e-6Proposed cut-off values< 20%***< 70%***<550 ARU¤<55%2< 90%2< 602Sensitivity100 %84,4 %100 %82,2 %82,9 %62,2%Specificity95,6 %77,8 %95,6 %86,7 %75,8 %82,2%* 95% confidence intervals; **paired t-test, ***as suggested by Gum et al, ¤as suggested by manufacturer, 2best fit on ROC curve

Intrauterine use of hyperconcentrated platelet concentrates collected with Trima Accel in a case of neonatal alloimmune thrombocytopenia

Due to the threat of serious or fatal bleedings, fetuses with neonatal alloimmune thrombocytopenia (NAIT) may need intrauterine platelet (PLT) transfusions. To prevent a volume overload or an ABO minor mismatch, standard PLT concentrates need to be washed to increase the PLT concentration and to reduce the plasma content. Hyperconcentrated single-donor PLT concentrates (HCPs) are a therapeutic alternative. The first case of NAIT successfully treated with HCPs collected with the Trima Accel (TA; Gambro BCT) is reported. A 31-year-old woman with a history of NAIT in the preceding pregnancy underwent cordocentesis three times during her third pregnancy (30th, 31st, and 32nd weeks of gestation). NAIT was confirmed by marked fetal thrombocytopenia, a maternal anti-human PLT antigen (HPA)-1a-immunoglobulin G (titer 1:128), and the appropriate HPA genotype of the fetus and the parents. On each cordocentesis procedure, a distinct volume of a HPA-1a-negative HCP with a PLT concentration of 3 x 10(6) PLTs per microL was transfused resulting in high corrected count increments after 2 hours. The HCPs were transfused within 10 hours after collection. One day after the last cordocentesis procedure, a cesarean section was performed. The newborn did not show any bleeding signs, and the PLT count remained on normal levels and no further PLT transfusions were needed. HCPs collected with TA are a useful alternative to washed standard PLT concentrates without the need for further manipulation of the product after collection. Further in vitro and in vivo studies are needed, however, to make definite recommendations for the shelf life of these HCP.

To adjust or not to adjust the platelet count in light transmission aggregometry in patients receiving dual aspirin/clopidogrel treatment

We evaluated whether the results of light transmittance aggregometry (LTA) differ when “native” platelet-rich plasma (PRP) or adjusted (to a standard platelet count of 250.000/µL) PRP is used in patients on dual antiplatelet therapy with aspirin and clopidogrel. LTA has been performed on the blood of 142 stable angina pectoris patients who were adequately pretreated with aspirin and clopidogrel. Platelet aggregation was significant higher in native PRP as compared to platelet count adjusted PRP (P<0.0001) for all four concentrations of adenosine-5′-diphosphate (ADP) (2, 5, 10 and 20 μmol/L). The interindividual variability was significantly higher in platelet count adjusted PRP as compared to native PRP when stimulated with 10 and 20 μmol/L of ADP. The absolute magnitude of aggregation in non-adjusted PRP is clearly dependent on platelet number. These observations are important since several studies have used empirically defined cut-off levels to segregate non-responders from responders to clopidogrel therapy.

Nouveau container de prélèvement : rôle dans la réduction de la contamination bactérienne des unités plaquettaires standard

Background Bacterial contamination of unstable blood products constitutes today the most frequent infectious risk transmitted by blood transfusion. Platelet concentrates are often incrimineted. As responsible germs are in general of cutaneous origin, a sample procedure with diversion of the first 20 ml during blood donation is studied. The aim of this study is to evaluate the results of this technique on bacterial contamination rate of standard platelet units prepared at the regional blood transfusion center in Casablanca. Study design and methods A comparative study of two types of sample pockets is made: 500 Standard Platelet concentrates (CPS) are prepared after collection using standard triple bags (Baxter) (group 1) and 560 pockets of CPS were prepared after collection using triple bags with Sample Diversion Pouch sampling system for elimination of the first 20 ml of donation (Macopharma and Terumo) (group 2). The skin was disinfected in two times with alcohol 70%. The bacteriological study was made in the two groups at the third day of conservation. Results Six CPS of group 1 were contaminated, of which five were staphylococci coagulase negative and one bacillus sp. Six CPS of group 2 were contaminated, of which five were staphylococci coagulase negative and one staphylococcus aureus. The bacteria isolated were those of cutaneous flora at 100%. Diversion of first 20 ml of blood donation results in a 16.6% reduction in bacterial contamination of CPS ( P > 0.05). Conclusion The non-significant reduction in the prevalence of the bacterial infection of CP formulates the problem of the indication of the sampling devices with derivation of first 20 ml during blood collection.

Microparticles Are a Major Determinant of Thrombin Generation Measured by Technothrombin®TGA.

We have recently shown that a thrombin generation assay using low concentrations of phospholipids (3,2 mM; 20% PS) can discriminate patients with an increased risk for recurrent venous thromboembolism after cessation of anticoagulant therapy from those with a lower risk (Hron et al JAMA 2006). Patients with peak thrombin of ≥400nM had a ~3-fold higher relative risk of recurrence than those with peak thrombin <400nM. This difference was not explained by a larger proportion of patients with clotting factor abnormalities among those with high peak thrombin. We therefore investigated which other factors might influence peak thrombin generation. For this purpose 35 healthy male plasma donors were analyzed. Thrombin generation was measured in standard platelet poor plasma and in plasma made microparticle (MP) free by centrifugation for 120 minutes at 35,250 x g using low (3,2μM) and high (32μM ) phospholipids concentrations at the same tissue factor concentration (71,6 pM). For control purposes, peak thrombin was measured under similar conditions in 12 individuals each having either heterozygous FVLeiden mutation, being on vitamin K-antagonist therapy (VKA; mean INR=3.2) or having a lupus anticoagulant. In healthy plasma donors peak thrombin as generated by tissue factor and low phospholipids (RCL) in platelet poor plasma was 110±40 nM and increased to 306±99 nM in the presence of high phospholipids (RCH). When MP free plasma was used, however, peak thrombin dropped to 23±13 nM (RCL) and 109±43 nM thrombin (RCH), respectively. When MPs were re-added to MP-free plasma samples, a dose dependent increase in peak thrombin was found both for RCL and RCH reagents. At a MP concentration of 25,000 added per 1 ml MP free plasma, peak thrombin was 381±24 nM for RCL and 399±63 nM for RCH. The effects of MPs on peak thrombin using RCL were much more pronounced than the differences in peak thrombin seen between plasmas from individuals with FVLeiden, during anticoagulant therapy or with lupus anticoagulant as compared to controls (FVLeiden : 161±50 nM; VKA: 50±12 nM; lupus: 37±23 nM; controls: 110±40 nM). These results indicate that MPs contained in platelet poor plasma are a major determinant of peak thrombin in thrombin generation assays when low phospholipids are used and might cause increased peak thrombin levels found in patients with recurrent venous thromboembolism.

Aspirin and Clopidogrel Resistance

Despite aspirin's and clopidogrel's proven benefit in reducing cardiovascular (CV) events, recurrent CV events still occur in patients receiving antiplatelet therapy. Many of these patients are resistant or only partially responsive to the antiplatelet effects of aspirin and clopidogrel, as determined by standard platelet assays. However, current clinical guidelines do not support routine screening for aspirin or clopidogrel resistance, in part because determination of the most appropriate screening test has not been established. This review attempts to (1) describe the phenomena of clinical aspirin and clopidogrel resistance (ie, treatment failure), (2) discuss the complexity of defining and identifying aspirin and clopidogrel resistance, (3) identify factors that may be responsible for aspirin and clopidogrel resistance, (4) outline several standard platelet function assays and their limitations, and (5) describe potential new antiplatelet therapies that may benefit aspirin- or clopidogrel-resistant patients.

The combined effect of platelet storage media and intercept pathogen reduction technology on platelet activation/activability and cellular apoptosis/necrosis: Lisbon-RBS experience

Platelets are known to undergo shape change, activation, a release reaction and apoptosis/necrosis during processing and storage, all of which are collectively known as the platelet storage lesion. Any additional processing may have some deleterious impact on platelet activability and functional integrity, which need to be investigated. This preliminary investigation was undertaken to establish the combined effects of standard platelet storage media and the intercept pathogen reduction technology on platelet activation and activability during 7 day storage, using buffy-coat derived platelets in standard storage media containing 35% plasma ( N = 24). P-selectin (CD62p) expression, a classical marker of platelet activation, and phosphatidylserine (PS) exposure on the platelet surface membrane, a hallmark of cellular necrosis/apoptosis, were both measured by flow cytometry. The results reveal significant increases in activation, from an average of 22.7% on day 1 before treatment to 31.6% on day 2 after treatment and 58.7% at the end of storage. Concomitantly, the basal expression of PS was slightly increased from 1.9% to 2.8% at day 2 after treatment and 7.3% at the end of storage. However, the functional reserve of platelets during storage, which reflects their capability to undergo activation and the release reaction when platelets were challenged with either calcium ionophore or thrombin, was relatively well maintained. These preliminary data confirm the earlier data on the use of intercept, and for the first time, based on the assessment of platelet functional integrity, suggest that platelet functional reserve is relatively well maintained, with little change in the formation of apoptotic cells.

P-Selectin Expression, but not GPIIb/IIIa Activation, is Enhanced in the Inflammatory Stage of Takayasu's Arteritis

Inflammation and thrombosis are closely related processes, but the association between disease activity and thrombogenicity in Takayasu's arteritis (TA) is poorly understood. To investigate the link between platelet activation and disease activity, flow cytometric analyses of platelet P-selectin and activated GPIIb/IIIa expression were performed in patients with TA. Twenty-two patients with TA, classified into active (Group A, n = 9) and inactive (Group I, n = 13) according to blood-derived inflammatory markers, and 14 healthy age- and gender-matched controls (Group C) were studied. Compared with Group C, the mean fluorescence intensity of P-selectin in response to 0.1-10 micromol/L of ADP was significantly upregulated in Group A, but not in Group I. No differences in platelet GPIIb/IIIa expression in stimulated platelets were seen among the 3 groups. Standard platelet aggregation studies revealed that disease activity did not influence platelet aggregation by ADP. P-selectin expression, but not activated GPIIb/IIIa, is enhanced in ADP-activated platelets from patients in the inflammatory stage of TA. P-selectin may play a significant role in the inflammatory and thrombotic responses associated with intractable TA, presumably by inducing platelet-leukocyte interactions.

Hyperconcentrated (Dry) Versus Standard Platelet Apheresis: An In Vitro Quality Study.

Introduction: Platelet concentrates (PC) collected by apheresis are effective in supporting deeply thrombocytopenic patients. The reduced risk of multiple allogeneic exposure and transmissible infectious diseases together with the high WBC depletion and diminished transfusion reactions are the main advantages offered by PC transfusion. At moment, the availability of several synthetic solutions for platelets storage permits to prepare hyperconcentrate(dry) apheresis platelets with the advantage of reducing febrile non-hemolytic transfusion reactions and, in low body weight patients the citrate toxicity, without the necessity of further manipulations. The aim of this study was to test the quality of 20 dry platelets (DP) in comparison to 20 standard plateletpheresis (SP) concentrates.Materials and methods: A total of 40 apheresis procedures were performed by the single-needle Cobe Trima separation device (Gambro BCT, Lakewood, CO, USA) collecting either DP or SP concentrates. Within 1h after collection, the bag containing DP was added with the appropriate amount (70% of DP final volume) of synthetic solution for platelets storage (SSP, MacoPharma). Both DP and SP concentrates were stored at room temperature with gentle agitation for 4 days. For both concentrates, platelet yield was calculated and in vitro studies of membrane glycoproteins expression and aggregation at day +1 and day +4 were carried out.Results: The comparison between 20 DP and 20 SP concentrates in terms of ability to aggregate in vitro and membrane glycoproteins expression at day +1 and day +4 of storage is reported in table A and B respectively.Conclusions: The in vitro tests documented a major activation of dry platelets. In particular, the ability to aggregate was reduced in the 20 DP concentrates analised and this phenomenon was more evident at day +4 of storage. The alteration of membrane glycoproteins expression (markers of storage lesion) confirms the lower in vitro quality of DP concentrates. The effectiveness of this new blood component in vivo should be evaluated in a controlled clinical trial.Table AAt collectionDay +1Day +4SPDPSPDPSPDPCollagen μg/ml 4938897828053ADP 10 μM32112416165Ristocetin 1.5 mg/ml919777817152Collagen 10 μg/ml + Adrenaline 10 μM989593949380ADP 10 μM + Adrenaline 10 μM877276615730Table BAt collectionDay +1Day +4SPDPSPDPSPDPGPIb alfa (MFI)5.065.756.316.135.264.54GPIIb-IIIa (MFI)35.4736.7134.6137.731.7640.9GP IV (MFI)11.4911.411.6710.2811.6511.38GP 5324.2327.1121.7425.9121.4631.84GMP 14021.7929.2922.6530.3820.5834.89