Endogenous glutathione and platelet function in platelet concentrates stored in plasma or platelet additive solution

Platelet function was studied in platelet concentrates by assay of the thrombin-induced release of endogenous serotonin and presence of the swirling phenomenon in relation to endogenous glutathione (GSH) and cysteine. In platelets stored in plasma, addition of cysteamine resulted in only a moderate fall in GSH after 5 days of storage, from an average of 14.91 to 11.46 nmol per 109 platelets. Exogenously added GSH had no effect, and addition of buthionine sulfoximine (BSO) resulted in almost complete depletion of GSH, to an average of 0.65 nmol per 109 platelets. Addition of cysteamine or GSH resulted in increased endogenous cysteine whereas BSO had no effect. In platelets stored in a platelet additive solution (T-sol), complete depletion of GSH was found in the presence of cysteamine, GSH and BSO. Endogenous serotonin was unchanged during storage both in plasma and in additive solution (2.8 nmol per 109 platelets). Despite almost total depletion of endogenous GSH, the thrombin-induced release of serotonin after 5 days' storage was significantly affected only in the presence of BSO in platelets stored in additive solution (mean values 72.3% vs. 63.3% of endogeneous serotonin, P < 0.05). Similarly, addition of cysteamine or GSH had no significant effect on swirling but BSO reduced the swirling score after 5 days' storage in platelet additive solution compared with plasma. After 10 days' storage, there was a significant reduction in swirling in the concentrates where BSO was added (P < 0.05).     

Blood platelet kinetics and platelet transfusion

The discovery of citrate anticoagulant in the 1920s and the development of plastic packs for blood collection in the 1960s laid the groundwork for platelet transfusion therapy on a scale not previously possible. A major limitation, however, was the finding that platelet concentrates prepared from blood anticoagulated with citrate were unsuitable for transfusion because of platelet clumping. We found that this could be prevented by simply reducing the pH of platelet-rich plasma to about 6.5 prior to centrifugation. We used this approach to characterize platelet kinetics and sites of platelet sequestration in normal and pathologic states and to define the influence of variables such as anticoagulant and ABO incompatibility on post-transfusion platelet recovery. The “acidification” approach enabled much wider use of platelet transfusion therapy until alternative means of producing concentrates suitable for transfusion became available.The identification of platelets as a distinct cellular element of blood with a critical role in hemostasis in the late 1800s (1) inevitably led to speculation about platelet transfusion as a treatment for bleeding in patients with thrombocytopenia. The realization of this goal was delayed for many years by technical barriers. Development of citrate-based anticoagulants in the 1920s and flexible plastic blood containers in the 1950s–1960s made it feasible to collect blood in a plastic pack containing standard acid-citrate-dextrose (ACD) anticoagulant, centrifuge it slowly, and express the supernatant platelet-rich plasma (PRP) into a plastic side-pack for convenient transfusion. Early studies showed that platelets from multiple units of blood were needed to achieve a therapeutic effect in a bleeding patient. To prevent volume overload, this required that platelets be concentrated before being transfused. The obvious way to accomplish this was to centrifuge PRP at high speed, remove the supernatant plasma, and suspend the pelleted platelets in a small volume by gently massaging the plastic pack. It soon became apparent that concentrates prepared in this way almost invariably contained large and small platelet aggregates and few single platelets. Not surprisingly, clinicians were reluctant to transfuse these preparations. It was known at this time that platelets isolated from blood that had been anticoagulated with EDTA could be pelleted from PRP by centrifugation and dispersed without difficulty. To meet the growing demand for platelet transfusions, the Fenwal Company developed the “EDTA Platelet Pack,” consisting of a plastic collection bag containing EDTA and an attached satellite bag into which PRP could be expressed, concentrated by centrifugation into a pellet, and suspended in a small volume of plasma. Red cells were returned to the donor to enable repeated platelet donations. Despite the obvious limitations of this approach, thousands of pooled EDTA platelet concentrates were transfused in the late 1950s and the 1960s. This procedure was labor intensive, and its application was restricted to relatively few, critically ill patients.In 1961, Gardner and associates conducted seminal studies to define the pathophysiology of various thrombocytopenic disorders (2, 3). They labeled EDTA platelets with NaCr⁵¹O₄ to follow the cells after transfusion. In these studies, very few labeled platelets were detected in the peripheral blood during the first few hours after transfusion. After this time, a variable number of cells reentered circulation. The immediate sequestration of a large fraction of the transfused cells, possibly in the liver and lung (4), followed by the eventual return of some platelets into circulation was considered to be a consequence of the labeling procedure. At this time, working at the Thorndike Memorial Laboratory of the Boston City Hospital, we were similarly interested in studying platelet kinetics, and we confirmed the findings of Gardner and coworkers about the circulation kinetics of EDTA platelets. We examined PRP prepared from EDTA and ACD whole blood under phase microscopy and noted that platelet morphology was quite different in the two preparations. In ACD preparations, platelets were discoid in shape, but in EDTA preparations, they assumed an irregular, almost spherical configuration. Another striking difference was the appearance of PRP examined in a light beam while being gently agitated: ACD platelets shimmered and swirled, whereas an EDTA platelet suspension was uniform in appearance throughout. We wondered whether structural changes induced in platelets by EDTA explained the failure of most of these platelets to circulate after transfusion and carried out studies to determine whether platelet clumping in concentrates from ACD-prepared PRP could be prevented. Evaluation of several variables revealed that when the pH of ACD PRP was reduced from its starting value of about 7.2 to about 6.5 before centrifugation, the pelleted platelets could readily be dispersed, yet retained their normal discoid shape. In whole blood or in PRP, this degree of “acidification” could be achieved by simply adding an extra quantity of ACD, the anticoagulant then used routinely for blood collection. The apparent benefit of acidification persisted through repeated centrifugations and made it possible to characterize recovery and survival of ACD platelets in normal subjects (5). Our studies revealed that about 75% of the labeled ACD platelets were recovered in the recipient immediately after transfusion (Figure ​(Figure1).1). After the initial transfusion, the presence of labeled platelets in the blood steadily declined over nine days. In contrast, labeled EDTA platelets peaked in the blood around one day after transfusion and steadily declined afterward (Figure ​(Figure1).1). Scanning of body organs with a directional scintillation counter revealed that most of the radioactivity from ACD platelets not recovered in the blood was initially present in the spleen; however, transfused EDTA platelets mainly concentrated in the liver. As ACD platelets were cleared from the circulation, Cr⁵¹ accumulated in the liver and spleen, indicating that these organs are the major sites of platelet deposition. The linear clearance pattern suggested that under normal circumstances, platelets die as a consequence of “senescence,” rather than being randomly utilized (5). Open in a separate windowFigure 1Survival of autologous “citrate platelets” after transfusion to a normal subject.Approximately 75% of labeled platelets were recovered in the circulation immediately after being transfused. The red area denotes the range of blood platelet radioactivity after the injection of Cr⁵¹-labeled “EDTA platelets” on 10 occasions in 7 normal subjects. Adapted from ref. 5. Freireich and his colleagues at the National Cancer Institute soon confirmed the superiority of platelet concentrates prepared from acidified ACD blood in producing sustained platelet increases in thrombocytopenic patients (6). Over the next few years, this simple maneuver facilitated much wider use of platelet transfusions, especially in patients being treated for hematologic malignancies. We used the new methodology to characterize platelet clearance and sites of sequestration in normal individuals (5, 7) and in patients with platelet destruction mediated by alloantibodies (8) and autoantibodies (9), as well as to more fully define the role of anticoagulants and ABO incompatibility on recovery and survival of transfused platelets (10). We also demonstrated that “hypersplenic” thrombocytopenia is largely caused by pooling of a significant fraction of the total circulating platelet mass in an enlarged spleen, rather than being a consequence of suppressed platelet production or premature platelet destruction (11). Although acidifying citrated blood or PRP to prepare platelet concentrates for transfusion represented a significant improvement over what was previously possible, other advances soon followed. Mourad found that platelet concentrates prepared from nonacidified ACD blood could be manually suspended with little clumping, provided the platelet pellet was allowed to rest for some time at room temperature before manipulation (12). Other key developments were the finding by Murphy et al. that platelet viability is best maintained by storage at room temperature (13) and the evolution of pheresis systems for isolating large quantities of platelets from single donors. To my knowledge, reversible aggregation of platelets pelleted from citrated PRP is still not fully understood, but it seems almost certain that fibrinogen binding to partially activated α_IIbβ₃ integrin (GPIIb/IIIa) is involved, since fibrinogen-dependent platelet aggregation is markedly inhibited at pH 6.5 (6, 14). The EDTA-induced structural changes in platelets were well characterized by White (15). The “swirling” of platelets was shown to be a consequence of their normal discoid shape and to correlate fairly well with post-transfusion viability (16).     

A flow cytometric method for platelet counting in platelet concentrates

BACKGROUND: The platelets (PLTs) in PLT concentrates are counted with hematology analyzers, but varying results among different hematology analyzers are observed, making comparisons very difficult. Due to the absence of red blood cells in PLT concentrates, the International Council for Standardization in Hematology (ICSH) reference method was modified to be used for PLT concentrates and validated in an international comparative study. STUDY DESIGN AND METHODS: Five PLT samples were shipped to eight participating centers of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative and counted on the same day. PLTs were stained with fluorescein isothiocyanate–labeled anti‐CD41a in tubes (TruCount, BD Biosciences), measured on a flow cytometer, and analyzed with a uniform template. These samples were also counted on 15 hematology analyzers. RESULTS: The ICSH method and newly developed BEST method yielded PLT counting results with less than 1% difference (not significant). The intercenter coefficient of variation (CV) of the BEST method was on average 6.3% versus 7.6% on average for hematology analyzers. The CV of individual hematology analyzers was on average 0.9%, which was considerably lower than for the flow cytometers with a mean of 3.7%. CONCLUSION: The BEST flow cytometric method has a smaller intercenter CV and a smaller center‐to‐center deviation from the group mean compared to hematology analyzers. Conversely, individual hematology analyzers are more precise than the flow cytometric method. Thus, the flow cytometric method provides a calibration tool to allow comparisons between centers, but there is no need to replace routine counting with hematology analyzers.     

Testing platelet mass versus platelet count to guide platelet transfusions in the neonatal intensive care unit

BACKGROUND: Platelet (PLT) transfusions can bestow significant benefits but they also carry risks. This study sought a safe means of reducing PLT transfusions to neonatal intensive care unit (NICU) patients with thrombocytopenia by comparing two transfusion guidelines, one based on PLT count and the other on PLT mass (PLT count times mean PLT volume).
STUDY DESIGN AND METHODS: Using a prospective, two-centered, before versus after design, PLT transfusion usage and hemorrhagic events were contrasted during a period when PLT count–based transfusion guidelines were in use (Period 1) versus a period when PLT mass–based guidelines were in use (Period 2).
RESULTS: No differences were observed between Periods 1 and 2 in NICU admissions, sex, race/ethnicity, percentage of inborn patients, or percentage of patients with a PLT count less than 50 × 10⁹ or 51 × 10⁹ to 99 × 10⁹/L. In the first period 3.6% of NICU admissions received one or more PLT transfusions. This fell to 1.9% during the second period (p < 0.002). The number of PLT transfusions administered per transfused patient was the same in both periods: 2.0 (1-23) (median [range]) in Period 1 and 2.0 (1-17) in Period 2 (p > 0.40). Significantly fewer PLT transfusions were given in Period 2 for prophylaxis (patient not bleeding; p < 0.001 vs. Period 1). The number given for bleeding did not change between the two periods. In Period 2 no increases were seen in rate of intraventricular hemorrhage (IVH); Grade 3 or 4 IVH; or pulmonary, gastrointestinal, or cutaneous bleeding.
CONCLUSIONS: The use of PLT mass–based NICU transfusion guidelines was associated with fewer PLT transfusions and no recognized increase in hemorrhagic problems.     

Integrins in platelet activation

Summary.  Heterodimeric receptors of the β1 and β3 integrin families mediate platelet adhesion and aggregation in hemostasis and thrombosis. In resting platelets, integrins are expressed in a low-affinity state but they shift to a high-affinity state and efficiently bind their ligands in response to cellular activation. This review summarizes recent advances in understanding the functional regulation and (patho-) physiological significance of individual platelet integrins with a special focus on studies in genetically modified mice. It is now recognized that β1 and β3 integrins have partially redundant roles in the adhesion process and that their activation is regulated by similar mechanisms, involving Ca²⁺-dependent and -independent signaling events and essential functions of talin-1 and kindlin-3 in the terminal activation step.     

Flow cytometric platelet cross-matching to predict platelet transfusion in acute leukemia

A great variety of patient- and product-related factors influence the outcome of platelet transfusions. Our study assessed the predictive value of a flow cytometric platelet cross match test for the outcome of HLA matched and unmatched platelet transfusions in patients with acute leukemia. Thirty nine patients (26 adults and 13 children) received 60 ABO compatible apheresis platelet unites ranging from 1 to 4 per patient (mean = 1.54; median = 2). We performed flowcytometric platelet cross-matching, HLA Class I typing by sequence-specific primer (SSP) for patients and complement-dependent cytotoxicity (CDC) for donors and screening of HLA Class I antibodies by ELISA. Effectiveness of platelet transfusion was evaluated using the corrected count increment which was calculated at 60 min and 18- to 24-h posttransfusion. Multivariate analysis was performed to detect which variable can predict transfusion response more than others. Cross-matched platelet transfusions associated with good response in 51.4% of transfusion events in adults and 73.3% in children. The noncrossmatched platelet transfusions associated with poor response in 83.3% in adults and 100% in children (P-values 0.143, 0.041, respectively). In the presence of clinical factors or HLA alloimmunization in adults, cross-matched platelets were associated with good response in 29.6 and 22.2% respectively. In children this occurred in 81.8 and 66.7%, respectively. In presence or absence of HLA matching, flow cytometry platelet cross-matching was the most predictor for transfusion response (P = 0.05). Because of the difficulties to find frequent HLA matched donors for acute leukemia patients; flow cytometric platelet cross-matching may provide the most useful way for selecting donors. It is useful even in the presence of alloimunization in children.     

Apheresis donors and platelet function: inherent platelet responsiveness influences platelet quality

BACKGROUND: Process-induced platelet (PLT) activation occurs with all production methods, including apheresis. Recent studies have highlighted the range and consistence of interindividual variation in the PLT response, but little is known about the contribution of a donors' inherent PLT responsiveness to the activation state of the apheresis PLTs or the effect of frequent apheresis on donors' PLTs. STUDY DESIGN AND METHODS: The relationship between the donors' PLT response on the apheresis PLTs was studied in 47 individuals selected as having PLTs with inherently low, intermediate, or high responsiveness. Whole-blood flow cytometry was used to measure PLT activation (levels of bound fibrinogen) before donation and in the apheresis PLTs. The effects of regular apheresis on the activation status of donors' PLTs were studied by comparing the in vivo activation status of PLTs from apheresis (n = 349) and whole-blood donors (n = 157), before donation. The effect of apheresis per se on PLT activation was measured in 10 apheresis donors before and after donation. RESULTS: The level of PLT activation in the apheresis packs was generally higher than in the donor, and the most activated PLTs were from high-responder donors. There was no significant difference in PLT activation before donation between the apheresis and whole-blood donors (p = 0.697), and there was no consistent evidence of activation in the donors immediately after apheresis. CONCLUSION: The most activated apheresis PLTs were obtained from donors with more responsive PLTs. Regular apheresis, however, does not lead to PLT activation in the donors.     

XT-1800i检测72份相关参数不全的血小板结果和直方图分析

目的 分析血小板相关参数不全的血小板直方图变化,判断血小板计数的准确性,追寻原因,探求解决方法.方法 对72份XT-1800i检测无血小板相关参数结果的标本进行显微镜计数,将两种方法的血小板计数结果进行统计学处理,并针对不同原因纠正.结果 根据血小板直方图的变化及原因可将72份标本分为4组,每组标本的血小板计数结果两种...     

A role for glycosphingolipid-enriched microdomains in platelet glycoprotein Ib-mediated platelet activation

BACKGROUND: Glycoprotein (GP) Ib, a platelet von Willebrand factor (VWF) receptor, plays a crucial role in thrombosis and hemostasis. As recent reports have suggested that GPIb partially locates in a particular region, designated as glycosphingolipid-enriched microdomains (GEMs), we hypothesized that GEMs play a central role in GPIb-mediated platelet activation. METHODS: Platelets were stimulated by VWF/botrocetin to activate platelets through GPIb. GEMs and non-GEMs were isolated by sucrose density gradient ultracentrifugation and the location of signaling molecules characterized. The role of GEMs-mediated signaling in platelet behavior was tested by platelet aggregation and by platelet interaction with immobilized VWF under flow conditions when GEMs were disrupted by methyl-beta-cyclodextrin (MbetaCD). RESULTS: GPIb was partially translocated to GEMs upon VWF/botrocetin stimulation. Immunoprecipitation of GPIb in GEMs and non-GEMs revealed that the tyrosine kinases, Src and Lyn, were associated with GPIb only in GEMs after GPIb-stimulation, and not in non-GEMs. Activation of PLCgamma2 was more intense in GEMs than non-GEMs. Disruption of GEMs by MbetaCD strongly inhibited tyrosine phosphorylation of Syk and PLCgamma2. Functional studies revealed that stable adhesion of platelets to a VWF-coated surface under flow was impaired by GEM disruption by MbetaCD. CONCLUSION: The combined results suggest that GEMs play an important role in GPIb-mediated platelet activation.     

Paroxetine decreases platelet serotonin storage and platelet function in human beings

BACKGROUND: Serotonin is a platelet agonist and potent vasoconstrictor that has recently received attention concerning its potential role in acute coronary artery thrombosis. Selective serotonin-reuptake inhibitors, such as paroxetine, are widely used antidepressant agents. We sought to characterize the potential inhibitory effect of paroxetine on platelet function. METHODS: Healthy male volunteers received 20 mg/d paroxetine for 2 weeks in a randomized, double-blind, placebo-controlled, two-way cross-over trial. RESULTS: Paroxetine decreased intraplatelet serotonin concentrations by -83% (P < .01). This inhibited platelet plug formation as reflected by a 31% prolongation of closure time measured with the platelet function analyzer-100 (P < .05). Furthermore, paroxetine lowered expression of the platelet activation marker CD63 in response to two different concentrations of thrombin receptor-activating peptide (P < .01). Plasma concentrations of prothrombin fragment, von Willebrand factor antigen, and circulating P-selectin remained unchanged in either period, indicating that paroxetine does not increase activation of coagulation, endothelium, or platelets in vivo, underlining a favorable safety profile. CONCLUSIONS: Paroxetine substantially decreases intraplatelet serotonin content and thereby reduces platelet plug formation under shear stress, and responsiveness to thrombin receptor activating peptide-induced platelet activation. Further studies will reveal whether these pharmacodynamic effects can be exploited for treatment of thrombotic artery disease.