Comparison of the platelet activation status of single-donor platelets obtained with two different cell separator technologies.

BACKGROUND: The microparticle content (MP%) of apheresis platelets-a marker of platelet activation-is influenced by donor factors and by external stressors during collection and storage. This study assessed the impact of apheresis technology and other factors on the activation status (MP%) of single-donor apheresis platelets. STUDY DESIGN AND METHODS: Data from six US hospitals that screened platelets by measuring MP% through dynamic light scattering (ThromboLUX) were retrospectively analyzed. Relative risks (RRs) were derived from univariate and multivariable regression models, with activation rate (MP% ≥15% for plasma-stored platelets; ≥10% for platelet additive solution [PAS]-stored platelets) and MP% as outcomes. Apheresis platform (Trima Accel vs Amicus), storage medium (plasma vs PAS), pathogen reduction, storage time, and testing location were used as predictors. RESULTS: Data were obtained from 7511 platelet units collected using Trima (from 16 suppliers, all stored in plasma, 20.0% were pathogen-reduced) and 2456 collected using Amicus (from four different collection facilities of one supplier, 65.0% plasma-stored, 35.0% PAS-stored, none pathogen-reduced). Overall, 30.0% of Trima platelets were activated compared to 45.6% of Amicus platelets (P < .0001). Multivariable analysis identified apheresis platform as significantly associated with platelet activation, with a lower activation rate for Trima than Amicus (RR: 0.641, 95% confidence interval [CI]: 0.578; 0.711, P < .0001) and a 6.901% (95% CI: 5.926; 7.876, P < .0001) absolute reduction in MP%, when adjusting for the other variables. CONCLUSION: Trima-collected platelets were significantly less likely to be activated than Amicus-collected platelets, irrespective of the storage medium, the use of pathogen reduction, storage time, and testing site.

Screening of red blood cells for extracellular vesicle content as a product quality indicator.

BACKGROUND: The controversy around the quality and clinical impact of stored and differentially manufactured red cell concentrates (RCCs) from different donor groups is ongoing. Current studies are limited by the lack of quality measures suitable for routine screening of RCCs. As extracellular vesicles (EVs) are markers of cellular activation or degradation, this study investigated the utility of EV screening to characterize the effects of RBCs production methods and storage. STUDY DESIGN AND METHODS: RCCs were prepared by whole blood filtration or red blood cell (RBC) filtration methods, centrifuged to prepare a supernatant, and tested for EV content (dynamic light scattering or tunable resistive pulse-sensing techniques), hemolysis, ATP, and RBC deformability on Days 7, 21, and 42 of storage. To simulate nondestructive quality control (QC) testing, 1 RBC unit was tested in parallel with six 10-mL aliquots that were stored in small-volume containers. RESULTS: EV content showed a linear increase with storage time (p < 0.001) and correlated with supernatant hemoglobin and inversely with ATP or RBC deformability. The method of component manufacturing influenced the characteristics of the EVs during storage. A strong correlation between both EV testing methods' measure of total EV was observed. EV content in the six aliquots were consistent at each time point but statistically higher than in the original RCCs on and after 21 days of storage. CONCLUSIONS: EV content correlates with measures of hemolysis and other RBC quality indicators and could be implemented as a routine screening tool for nondestructive QC testing of RCCs.

Routine Screening Method for Microparticles in Platelet Transfusions.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Microparticle detection to guide platelet management for the reduction of platelet refractoriness in children - A study proposal.

Microparticles have been shown to shed from a variety of viable cells as a consequence of inflammatory processes, activation or physical stress. Seventy to 90% of circulating microparticles are thought to be platelet-derived. The content of microparticles in blood collected from normal blood donors is highly variable and transfers into the final blood component. Elevated microparticle content (MPC) in donor blood might indicate an asymptomatic clinical condition of the donor which might affect the transfusion recipient, particularly pediatric patients. ThromboLUX is a new technology designed to routinely test biological samples for microparticle content. We compared MPC in platelet-rich plasma (PRP) of apheresis donors and the corresponding INTERCEPT-treated apheresis products (N=24). The MPCs in donor and product samples were correlated (r=0.74, P<0.001). Microparticles were significantly reduced after plasma replacement and INTERCEPT treatment. These findings are supported by phase contrast microscopy. Platelet transfusions given to patients with fever or systemic inflammation are less efficacious. In addition, transfusing heterogeneous platelets - concentrates with high MPC and activated platelets - to patients whose immune systems are activated might tip them over a threshold and cause platelet refractoriness. Restricting prophylactic platelet transfusions to homogeneous products - concentrates with resting platelets and therefore low MPC - may reduce the risk of refractoriness in cancer patients, especially children with immature immunity. To test this hypothesis we introduce an evaluation protocol for platelet management, i.e., keeping a split inventory of homogeneous and heterogeneous platelets, and using only homogeneous platelets for prophylaxis as a strategy to reduce refractoriness.

Microparticle content of platelet concentrates is predicted by donor microparticles and is altered by production methods and stress.

In circulation, shedding of microparticles from a variety of viable cells can be triggered by pathological activation of inflammatory processes, by activation of coagulation or complement systems, or by physical stress. Elevated microparticle content (MPC) in donor blood might therefore indicate a clinical condition of the donor which, upon transfusion, might affect the recipient. In blood products, elevated MPC might also represent product stress. Surprisingly, the MPC in blood collected from normal blood donors is highly variable, which raises the question whether donor microparticles are present in-vivo and transfer into the final blood component, and how production methods and post-production processing might affect the MPC. We measured MPC using ThromboLUX in (a) platelet-rich plasma (PRP) of 54 apheresis donors and the corresponding apheresis products, (b) 651 apheresis and 646 pooled platelet concentrates (PCs) with plasma and 414 apheresis PCs in platelet additive solution (PAS), and (c) apheresis PCs before and after transportation, gamma irradiation, and pathogen inactivation (N = 8, 7, and 12 respectively). ThromboLUX-measured MPC in donor PRP and their corresponding apheresis PC samples were highly correlated (r = 0.82, P = .001). The average MPC in pooled PC was slightly lower than that in apheresis PC and substantially lower in apheresis PC stored with PAS rather than plasma. Mirasol Pathogen Reduction treatment significantly increased MPC with age. Thus, MPC measured in donor samples might be a useful predictor of product stability, especially if post-production processes are necessary.

Could Microparticles Be the Universal Quality Indicator for Platelet Viability and Function?

High quality means good fitness for the intended use. Research activity regarding quality measures for platelet transfusions has focused on platelet storage and platelet storage lesion. Thus, platelet quality is judged from the manufacturer's point of view and regulated to ensure consistency and stability of the manufacturing process. Assuming that fresh product is always superior to aged product, maintaining in vitro characteristics should preserve high quality. However, despite the highest in vitro quality standards, platelets often fail in vivo. This suggests we may need different quality measures to predict platelet performance after transfusion. Adding to this complexity, platelets are used clinically for very different purposes: platelets need to circulate when given as prophylaxis to cancer patients and to stop bleeding when given to surgery or trauma patients. In addition, the emerging application of platelet-rich plasma injections exploits the immunological functions of platelets. Requirements for quality of platelets intended to prevent bleeding, stop bleeding, or promote wound healing are potentially very different. Can a single measurable characteristic describe platelet quality for all uses? Here we present microparticle measurement in platelet samples, and its potential to become the universal quality characteristic for platelet production, storage, viability, function, and compatibility.

Characterization of platelet concentrates using dynamic light scattering.

BACKGROUND: Each year, millions of platelet transfusions save the lives of cancer patients and patients with bleeding complications. However, between 10 and 30% of all platelet transfusions are clinically ineffective as measured by corrected count increments, but no test is currently used to identify and avoid these transfusions. ThromboLUX(®) is the first platelet test intended to routinely characterize platelet concentrates prior to transfusion. METHODS: ThromboLUX is a non-invasive, optical test utilizing dynamic light scattering to characterize a platelet sample by the relative quantity of platelets, microparticles, and other particles present in the sample. ThromboLUX also determines the response of platelets to temperature changes. From this information the ThromboLUX score is calculated. Increasing scores indicate increasing numbers of discoid platelets and fewer microparticles. ThromboLUX uses calibrated polystyrene beads as a quality control standard, and accurately measures the size of the beads at multiple temperatures. RESULTS: Results from apheresis concentrates showed that ThromboLUX can determine the microparticle content in unmodified samples of platelet concentrates which correlates well with the enumeration by flow cytometry. ThromboLUX detection of microparticles and microaggregates was confirmed by microscopy. CONCLUSION: ThromboLUX provides a comprehensive and novel analysis of platelet samples and has potential as a noninvasive routine test to characterize platelet products to identify and prevent ineffective transfusions.

Novel test for microparticles in platelet-rich plasma and platelet concentrates using dynamic light scattering.

BACKGROUND: The level and clinical importance of platelet (PLT)-derived microparticles (PMPs) in PLT-rich plasma (PRP) and PLT transfusions is largely unknown due to the lack of technology to routinely determine the number and size of PMP in PLT samples. Dynamic light scattering (DLS) is ideally suited to measure particles of submicron size but has previously been limited to the analysis of PLT-free samples. STUDY DESIGN AND METHODS: PMPs were enumerated in 81 PRP and 79 apheresis PLT concentrate (APC) samples from the same donors using ThromboLUX (LightIntegra Technology, Inc.), a new DLS PLT quality test. The ThromboLUX results were compared with flow cytometry. Phase contrast and differential interference contrast (DIC) microscopy were used to qualitatively determine PMP levels. RESULTS: The relative counts of PMPs measured by flow cytometry strongly correlated with the relative light scattering intensities of the PMP determined by ThromboLUX in both PRP (R = 0.7596, p < 0.0001) and APC (R = 0.6572, p < 0.0001) samples. High or low PMP levels in PLT samples were confirmed by phase contrast and DIC microscopy. The mean PMP radius measured with ThromboLUX, an absolute sizing technology, was 117.1 ± 77.6 nm as determined from the distribution of PMP content in all PLT samples investigated in this study. CONCLUSIONS: Correlation with flow cytometry and microscopy showed that ThromboLUX is well suited to measure PMP concentration and size distribution in PLT concentrate samples. In combination with noninvasive sampling, ThromboLUX could provide routine microparticle enumeration of PLT-containing samples.

Long-term aspirin and clopidogrel response evaluated by light transmission aggregometry, VerifyNow, and thrombelastography in patients undergoing percutaneous coronary intervention.

BACKGROUND: A reduced response to aspirin and clopidogrel predicts ischemic events, but reliable tests are needed to identify low responders. We compared 3 platelet-function tests during long-term dual treatment with aspirin and clopidogrel. METHODS: Patients who underwent a percutaneous coronary intervention and were receiving a combination of 325 mg/day aspirin and 75 mg/day clopidogrel were followed for 1 year. Blood was sampled 5 times during this period for 3 tests: light transmission aggregometry (LTA) assay, with 5.0 micromol/L ADP or 1.0 mmol/L arachidonic acid (AA) used as an agonist; VerifyNow assay, with the P2Y(12) or aspirin cartridge (Accumetrics); and thrombelastography (TEG), stimulated by 2.0 micromol/L ADP or 1.0 mmol/L AA. RESULTS: Twenty-six of 33 patients completed all scheduled visits. A low response to clopidogrel was found in a few patients at variable frequencies and at different visits, depending on the method and criteria used. We found a moderate correlation between the LTA (ADP) and VerifyNow (P2Y(12) cartridge) results, but the TEG (ADP) results correlated poorly with the LTA and VerifyNow results. A low response to aspirin was found with the VerifyNow (aspirin cartridge) and TEG (AA) methods on 6 and 2 occasions, respectively, but not with the LTA (AA) method, except for 1 occasion caused by probable noncompliance. CONCLUSIONS: Detecting a low response to clopidogrel depends largely on the method used. Which method best predicts ischemic events remains uncertain. A low response to aspirin is rare with AA-dependent methods used at the chosen cutoffs. In some patients, the response to clopidogrel or aspirin may be classified differently at different times, even with the same method.

Platelet quality measured with dynamic light scattering correlates with transfusion outcome in hematologic malignancies.

BACKGROUND: A clinically meaningful test for platelet (PLT) quality could improve the transfusion management of patients. The aim of this pilot study was to determine whether a new measure of PLT quality and function based on dynamic light scattering (DLS) correlates with transfusion outcome. STUDY DESIGN AND METHODS: For a total of 160 transfusions, the pretransfusion, 1 hour posttransfusion, and 24-hour posttransfusion PLT counts were routinely measured in 49 patients (31 male, 18 female; age 46 +/- 15 years) with hematologic malignancies. The corrected count increments (CCIs) at 1 hour (PLT recovery) and 24 hours (PLT survival) were calculated and used as the transfusion outcome measures. The ThromboLUX score (LightIntegra Technology, Inc., Vancouver, BC, Canada; range, 0-40; cutoff, 12) and the PLT morphology score of the PLT concentrates were determined and compared to transfusion outcome. RESULTS: The CCIs and ThromboLUX scores were normally distributed and showed a strong correlation (n = 96, in the mixed regression model the adjusted coefficient is R = 0.6292, p < 0.0001), while other variables such as product type, age, and microscopic PLT morphology score were not correlated with transfusion outcome (p > 0.05). Importantly, 12 of 96 transfusions with poor PLT quality were clinically ineffective, that is, did not adequately increase the PLT counts in the recipients. One patient died after receiving three consecutive ineffective PLT transfusions with a low ThromboLUX score. CONCLUSION: In this pilot study, the ThromboLUX score strongly correlated with transfusion outcome (PLT recovery and survival) independent of clinical and product issues.

The ELAPSE (Evaluation of Long-Term Clopidogrel Antiplatelet and Systemic Anti-Inflammatory Effects) study.

OBJECTIVES: This study was designed to evaluate the effects of long-term clopidogrel and aspirin administration on platelet aggregation, activation, and inflammation. BACKGROUND: Clopidogrel resistance was described in 15% to 30% of patients with short-term therapy, but its antiplatelet effects with long-term therapy is unknown. METHODS: We performed a prospective study of patients undergoing coronary stenting who were on aspirin for > or =5 days but not previously on clopidogrel. Clopidogrel 600 mg was given before stenting. Clopidogrel 75 mg/day and aspirin 325 mg/day were continued for 1 year. Light-transmittance aggregometry with 5-micromol/l adenosine diphosphate and 1-mmol/l arachidonic acid stimulation; VerifyNow clopidogrel and aspirin assays; platelet activation receptor expression of CD40L, CD62P, and PAC-1 (antibody against activated glycoprotein IIb/IIIa); and inflammatory markers of soluble CD40L and P-selectin, high-sensitivity C-reactive protein, interleukin-10, and interleukin-18 were measured at baseline; 1 day; and 1, 6, and 12 months. Our primary analysis compared light-transmittance aggregometry aggregation at 1 versus 12 months. RESULTS: We enrolled 26 patients who completed a 1-year follow-up. Maximal platelet adenosine diphosphate-stimulated aggregation was 61.8 +/- 25.9% at baseline, 22.1 +/- 18.3% at 1 day, 30.6 +/- 16.8% at 1 month, 29.0 +/- 13.3% at 6 months, and 26.7 +/- 13.6% at 12 months (p = 0.099 for 12 months vs. 1 month). VerifyNow clopidogrel platelet inhibition was similar at 12 months versus 1 month (38.9 +/- 19.7% vs. 45.6 +/- 26.7%, p = 0.578). Likewise, there was no difference in aspirin's effects on platelet aggregation at 12 months versus 1 month. In contrast, platelet activation receptor expression of CD40L, CD62P, and PAC-1 were higher at 12 months versus 1 month. CONCLUSIONS: Our pilot study showed no attenuation of clopidogrel's effects on platelet aggregation with long-term administration. However, platelet activation receptor expression increased with time and should be further evaluated.

Effects of aspirin and clopidogrel in healthy men measured by platelet aggregation and PFA-100.

There are no generally accepted definitions for low-response (frequently called resistance) to the platelet inhibitors, aspirin and clopidogrel. Low-response may increase the risk of cardiovascular events in atherosclerotic patients. We aimed to define the normal drug responses in healthy men. Platelet function was measured in 20 healthy men during 11 days of aspirin or clopidogrel intake, using light transmission aggregometry (LTA) and the Platelet Function Analyzer 100 (PFA-100). The lower limits for LTA at baseline were 64% and 61%, using arachidonic acid and ADP as agonists, respectively. During aspirin intake the LTA results were stable from day to day, and an upper limit of 9% arachidonic acid stimulated aggregation was found. Clopidogrel intake was best shown by ADP induced aggregation. However, two out of 20 individuals exhibited low-response to clopidogrel. In the remaining 18 volunteers an upper limit of 48% aggregation was found. We found an upper limit for collagen-epinephrine stimulated PFA-100 results of 166 s at baseline. During aspirin intake, these results varied considerably from day to day in nine out of 20 men, resulting in an overlap between the reference ranges at baseline and during therapy. In conclusion, platelet inhibition by aspirin and clopidogrel assessed by aggregometry was stable during 11 days of treatment and reference ranges were established. The PFA-100 results varied greatly and low-response was not precisely defined by this method.

Quebec platelet disorder: features, pathogenesis and treatment.

Quebec platelet disorder (QPD) is a rare, autosomal-dominant, inherited bleeding disorder that is associated with unique abnormalities in fibrinolysis. Its hallmark features are delayed-onset bleeding following hemostatic challenges that responds to fibrinolytic inhibitor therapy and increased expression and storage of the fibrinolytic enzyme urokinase plasminogen activator in platelets, without increased plasma urokinase plasminogen activator or systemic fibrinolysis. The increased urokinase plasminogen activator in QPD platelets is only partially inhibited, and, as a result, there is intraplatelet generation of plasmin, and secondary degradation of many platelet alpha-granule proteins. During clot formation, the urokinase plasminogen activator released by QPD platelets leads to platelet-dependent increased fibrinolysis, and this is postulated to be a major contributor to QPD bleeding. The focus of the present review is to summarize the current state of knowledge on QPD, including the history of this disorder, its clinical and laboratory features, and recommended approaches for its diagnosis and treatment.

Past and future approaches to assess the quality of platelets for transfusion.

No automated test exists to routinely measure platelet quality. Currently, the short, 5-day shelf life of platelet concentrates is largely dictated by the risk associated with bacterial contamination and not by platelet quality. With the implementation of bacterial testing and pathogen inactivation, platelet quality will become the major determinant for the shelf life of platelet concentrates. However, extended use of platelet concentrates stored beyond 5 days will require quality testing. In addition, high platelet quality would be expected to result in improved clinical efficacy, determined by count increment, improved hemostasis, and lower risk for adverse reactions in recipients. No in vitro quality test has yet demonstrated a good correlation with clinical efficacy or improved hemostasis. This review focuses on those tests of platelet quality that are based on platelet morphology. These include visual inspection of swirling, microscopic morphology score, measurement of light transmission through platelet concentrates, and platelet light scattering techniques. Recently, a new test for platelet quality has been introduced that uses dynamic light scattering. The advantages and remaining challenges for dynamic light scattering before it can become a routine platelet quality test are discussed.

Diagnosing platelet delta-storage pool disease in children by flow cytometry.

Bleeding problems are symptomatic of platelet delta-storage pool diseases (SPDs) such as Hermansky-Pudlak syndrome. Although at present no cure is available for delta-SPD, early diagnosis is of great importance for prophylactic and supportive treatment. This study tested the usefulness of a flow cytometric assay for platelet serotonin in children. The assay was used to diagnose delta-SPD in a 10-year-old girl. Platelet serotonin levels were significantly lower in the patient than in all healthy control subjects (10 children and 10 adults). The serotonin results were supported by traditional tests, which are transmission electron microscopy of whole mounts and adenosine triphosphate release by lumi-aggregometry. The flow cytometric serotonin assay is a major improvement to current pediatric diagnostics. The advantages of this test are small sample volume of fresh or fixed/frozen platelets, availability of objective results within 2 hours of obtaining the blood sample, and automated analysis by flow cytometry.

Platelet serotonin levels support depression scores for women with postpartum depression.

OBJECTIVE: It is very challenging to make an unbiased diagnosis of psychiatric illness. Platelets have long been proposed as easily obtainable, neurological models of serotonergic neurons. This study examined whether a new measurement for platelet serotonin could aid in the diagnosis of postpartum depression and support the results from questionnaires. METHODS: Study participants included 11 patients with postpartum clinical depression according to the Diagnostic and Statistical Manual of Mental Disorders, fourth edition, criteria. Blood was donated either at acute onset of depression before treatment (n = 5) or while patients were nonresponsive to paroxetine treatment (n = 8; 2 of these patients dropped out). A follow-up sample was donated approximately 8 weeks later during paroxetine treatment (n = 11). Platelet serotonin was determined with a new immunocytochemical assay and standard high-pressure liquid chromatography. Serotonin levels were compared with Hamilton Depression Rating Scale scores. RESULTS: Platelet serotonin levels in patients with depression before paroxetine treatment or nonresponsive to their initial paroxetine regimen were reduced to 50% of normal levels. Treatment-induced severe reduction of platelet-associated serotonin only occurred in responsive patients. Mean platelet serotonin levels were significantly lower in responders (17.3%, standard deviation [SD] 4%), compared with nonresponders (33.4%, SD 8%; p < 0.001). CONCLUSION: Platelet serotonin levels obtained with a new immunocytochemical test correlated well with results from depression scoring and might be useful as evidence-based support for questionnaires.

Portable dynamic light scattering instrument and method for the measurement of blood platelet suspensions.

No routine test exists to determine the quality of blood platelet transfusions although every year millions of patients require platelet transfusions to survive cancer chemotherapy, surgery or trauma. A new, portable dynamic light scattering instrument is described that is suitable for the measurement of turbid solutions of large particles under temperature-controlled conditions. The challenges of small sample size, short light path through the sample and accurate temperature control have been solved with a specially designed temperature-controlled sample holder for small diameter, disposable capillaries. Efficient heating and cooling is achieved with Peltier elements in direct contact with the sample capillary. Focusing optical fibres are used for light delivery and collection of scattered light. The practical use of this new technique was shown by the reproducible measurement of latex microspheres and the temperature-induced morphological changes of human blood platelets. The measured parameters for platelet transfusions are platelet size, number of platelet-derived microparticles and the response of platelets to temperature changes. This three-dimensional analysis provides a high degree of confidence for the determination of platelet quality. The experimental data are compared to a matrix and facilitate automated, unbiased quality testing.

The influence of selective serotonin reuptake inhibitors on human platelet serotonin.

Clinical depression has been proposed to be an independent risk factor for cardiovascular disease. While it is suggested that selective serotonin reuptake inhibitors (SSRIs) reduce the risk of acute cardiovascular problems of depressed patients, the effect of SSRIs on platelets, the only blood cells committed to serotonin (5-HT) transport, remains largely unknown. The goal of this pilot study was to measure the 5-HT levels in platelets of untreated and SSRI-treated depressed patients and normal subjects and to determine whether the interaction of SSRIs with platelets can explain their possible cardiovascular benefit in patients with depression. Platelet 5-HT was determined by an immunocytochemical assay and high-pressure liquid chromatography with electrochemical detection (HPLC-ECD). In normal control subjects without cardiovascular disease, 78 +/- 8% of platelets were 5-HT-positive (n = 14). Depression caused a significant reduction in platelet 5-HT to 46 +/- 21% in untreated patients (n = 13) and 22 +/- 13% in SSRI-treated patients (n = 14). As a class, all selective serotonin reuptake inhibitors significantly reduced the 5-HT concentration in patient platelets. An inverse relationship of 5-HT level and dose of medication might be suggested. These results correlated well with 5-HT data from HPLC (r = 0.8509, p < 0.001). SSRIs did not affect platelet aggregation and dense granule release in response to thrombin, but significantly reduced ADP-induced platelet aggregation and dense granule release in both patient and normal control samples. The active inhibition of platelet aggregation by SSRIs might explain their cardiovascular benefit.

A novel immunocytochemical assay for the detection of serotonin in platelets.

A method for the rapid, inexpensive and easy detection of platelet serotonin (5-hydroxytryptamine, 5-HT) is not currently available. Consequently, many patients suffering from unresolved platelet-related bleeding disorders are not examined for a possible platelet 5-HT deficiency. The direct measurement of 5-HT concentration with high-performance liquid chromatography (HPLC) or serotonin enzyme-linked immunosorbent assay (ELISA) is costly and highly demanding. Indirect methods, which determine the content of ATP or calcium with lumi-aggregometry or electron microscopy, rely upon the assumption that the ATP or calcium concentration is equivalent to that of 5-HT. We have developed a fluorescence-based assay for 5-HT that can be performed within 2 h on fresh or frozen samples using a fluorescence microscope or a flow cytometer. The assay requires only 0.2 ml of platelet-rich plasma and might therefore be of particular interest for paediatric patients. Samples from control and patient donors were analysed for 5-HT with the new immunocytochemical assay in comparison with HPLC and/or 5-HT ELISA. Patients with Hermansky-Pudlak syndrome were readily identified. The new assay was also reliable in cases where the 5-HT content of dense granules was not correlated with the calcium or ATP content, such as in calcium deficiency or in the presence of selective serotonin reuptake inhibitors.