Markers of platelet activation and apoptosis in platelet concentrates collected by apheresis.

BACKGROUND: A product with well-preserved haemostatic function of platelets is the ultimate goal of platelet concentrate production. However, platelet activation and apoptosis are induced by both collection and storage of platelet concentrates. AIM OF STUDY: Platelet concentrates obtained either by two blood separators with different technology of apheresis (Haemonetics MCS+, Haemonetics Corp. Braintree, USA and Trima Accel, Gambro BCT Inc., Lakewood, USA, respectively) or derived from buffy-coat were compared using evaluation of pH, LDH, lactate, glucose, annexin V, and sP-selectin levels immediately after collecting and at the end of expiration to estimate the differences in the activation and apoptosis of platelets in these products. RESULTS: The lowest degree of platelet activation was found in products obtained by Haemonetics MCS+ apparatus at the time of collection. Platelet concentrates obtained by apheresis revealed higher rise of LDH, annexin V and sP-selectin compared to buffy-coat derived platelets. Products from Haemonetics MCS+ showed higher rise of annexin V in comparison with products from Trima separator. Increase of LDH and sP-selectin in both apheresis products was comparable. CONCLUSIONS: On the basis of changes of sP-selectin and annexin V levels it could be concluded that initial platelet activation, which is induced by apheresis, is very likely without any further impact on quality of platelets during storage. Development of platelet storage lesions is influenced especially by storage conditions and platelet concentration in products.     

Comparison of platelet quality and function across apheresis collection platforms

Background

Platelet concentrates (PLT) can be manufactured using a combination of apheresis collection devices and suspension media (plasma or platelet additive solution (PAS)). It is unclear how platelet quality and hemostatic function differ across the current in-use manufacturing methods in the United States. The objective of this study was therefore to compare baseline function of PLT collected using different apheresis collection platforms and storage media.

Study Design and Methods

PLT were collected at two sites with identical protocols (N = 5 per site, N = 10 total per group) on the MCS® + 9000 (Haemonetics; “MCS”), the Trima Accel® 7 (Terumo; “Trima”), and the Amicus Cell Separator (Fresenius Kabi, “Amicus”). MCS PLT were collected into plasma while Trima and Amicus PLT were collected into plasma or PAS (Trima into Isoplate and Amicus into InterSol; yielding groups “TP”, “TI” and “AP”, “AI”, respectively). PLT units were sampled 1 h after collection and assayed to compare cellular counts, biochemistry, and hemostatic function.

Results

Differences in biochemistry were most evident between plasma and PAS groups, as anticipated. MCS and TP had the highest clot strength as assessed by viscoelastometry. AI had the lowest thrombin generation capacity. Both TP and TI had the highest responses on platelet aggregometry. AI had the greatest number of microparticles.

Discussion

Platelet quality and function differ among collection platforms at baseline. MCS and Trima platelets overall appear to trend toward higher hemostatic function. Future investigations will assess how these differences change throughout storage, and if these in vitro measures are clinically relevant.     

Differential induction of P-selectin expression on platelets by two cell separators during plateletpheresis and the effect of gender on the release of soluble P-selectin

BACKGROUND: Though a number of studies have elegantly characterized platelet activation during storage, less attention has been paid to the initial activation caused by different collection procedures. STUDY DESIGN AND METHODS: The effects of two blood cell separators on the initial activation of platelets were measured by flow cytometric analysis of P-selectin expression in 13 male donors on one cell separator (CS 3000 Plus) and 11 men and 9 women on the other (MCS 3P). In addition, the storage and release of soluble P-selectin (circulating P-selectin [cP-selectin]) by platelets were quantified, to determine whether the change in cP-selectin is a more sensitive marker for initial platelet activation, and the influence of gender on measured endpoints was evaluated. RESULTS: The CS 3000 Plus increased the percentage of P-selectin-positive platelets from a median of 3.4 percent before apheresis to 7.6 percent (p = 0.006) in platelet concentrates (PCs), whereas the MCS 3P did not (p = 0.002 between the two cell separators). When preapheresis cP-selectin levels were compared to those in apheresis PCs, cP-selectin increased from 51 to 101 ng per mL in plasma of CS 3000 Plus PCs, whereas cP-selectin levels increased from 53 to 78 ng per mL in MCS 3P PCs (men) and from 48 to 99 ng per mL in MSC 3P PCs (women) (p<0.005 for all). The relative increase in cP-selectin was higher in women than in men in MCS 3P PCs (p = 0.013). Concomitantly, the amount of P-selectin stored in platelets before apheresis decreased (p<0.025 for all). When donors undergoing apheresis on the MCS 3P were compared, the amount of P- selectin stored in the platelets of PCs was higher in men than women (p = 0.026). CONCLUSION: This trial shows 1) that initial activation of platelets obtained with the MCS 3P is less than that of platelets obtained with the CS 3000 Plus; 2) that the increase in cP-selectin is a more sensitive marker for initial platelet activation than the expression of P-selectin on the surface; and 3) that the relative amount of cP-selectin is higher in women than in men given the same stimulus. Differences in platelet activation by various cell separators and the sex of the donor may contribute to variability of PC quality.     

Cellular contamination of plasma collected with various apheresis systems.

Efforts to improve the purity of blood products have mainly focused on reducing white blood cell (WBC) levels in cellular blood products. Relatively little attention has been given to the cellular purity of plasma. We evaluated plasma units collected on six apheresis systems: Dideco Excel, Haemonetics-MCS+, Fresenius-AS-Tech 204, Baxter-Amicus, Gambro BCT COBE Spectra and Gambro BCT-Trima. Collected plasma volumes averaged 300-350 ml for the various systems. Plasma samples were analyzed for platelet (PLT) content (Technicon H3, Bayer) and residual WBC (Imagn 2000). Results are given below. Platelet levels were consistently low for MCS+, COBE Spectra and Trima (all <50 x 10(3) microl(-1)), and were highest with AS204. Residual WBC levels were relatively low in all systems except MCS+. Extremely low levels were observed in Trima plasma. All of the Trima and Spectra units contained <1 x 10(6) WBC per product. With Excel, AS-Tech 204 and Amicus, 1 to 2 units were found to have >1 x 10(6) WBC, while almost all units from MCS+ exceeded this limit. Different levels of plasma purity were obtained with different apheresis systems. The Gambro BCT COBE Spectra and Trima systems were found to achieve consistently low levels of both platelets and WBC.     

单采血小板储存样品中某些细胞因子含量的变化

本研究探明不同血细胞分离机所采集的单个供者血小板（single donor platelets，SDP）在储存期间细胞因子含量的变化。使用MCS^＋、Trima和Amicus3种血细胞分离机采集18份SDP，于血库标准条件下储存，于第1、3、5、7天取样检测储存期内白介素8（IL-8）、RANTES、CD154和肿瘤生长因子β1（TGF-β1）、血管内皮细胞生长因子（VEGF）等细胞因子含量的变化情况。结果显示：MCS^＋、Trima和Amicus机器采集的SDP，在储存期间随着时间的延长细胞因子IL-8、RANTES、CD154、TGF-β1及VEGF的含量逐渐增高，但MCS^＋机器采集SDP的IL-8的含量在保存期的增高水平，与Trima和Amicus法采集的SDP水平相比有显著性差异（P〉0．05）；而其余的细胞因子含量虽有增高，但无显著性差异．结论：单采血小板储存期间IL-8、RANTES、CD154、TGF-β1和VEGF等细胞因子的含量随保存时间的延长有升高的趋势；少白细胞的单采血小板中的IL-8表达相对较少。     

不同型号血细胞分离机对献血者细胞参数与机采血小板采集效率比较

目的比较不同型号血细胞分离机对献血者细胞参数与机采血小板采集效率。方法选取2018年1月至2019年11月我站的318例献血者,其中159名献血者应用AmiCORE、MCS+及Trima血细胞分离机捐献双份血小板,平均分为3组,另外159名献血者应用AmiCORE、MCS+及Trima血细胞分离机捐献单份血小板,平均分为3组。比较三组的单份及双份血小板细胞参数不合格率及分离机性能。结果MCS+组单份血小板细胞参数不合格率低于AmiCORE组和Trima组,差异具有统计学意义(P<0.05)。MCS+组单份血小板采集效率及时间高于AmiCORE组及Trima组,差异具有统计学意义(P<0.05);Trima组抗凝剂使用量及全血处理量多于MCS+组及AmiCORE组(P<0.05)。三组双份血小板细胞参数不合格率比较,差异均无统计学意义(P>0.05)。MCS+组双份血小板采集效率高于AmiCORE组及Trima组,差异具有统计学意义(P<0.05);AmiCORE组采集时间长于MCS+及Trima组,Trima组抗凝剂使用量及全血处理量多于MCS+及AmiCORE组,差异具有统计学意义(P<0.05)。结论AmiCORE、MCS+及Trima血细胞分离机对产品采集具有较高采集效率,符合国家有关标准,血液制备和采集过程可供,各机型血小板细胞参数不合格率、血小板分离机性能存在区别。血液制备及血液采集过程具有可控性,应依据献血者自身特点选择不同血细胞分离机。     

Quality of packed red blood cells and platelet concentrates collected by multicomponent collection using the MCS plus device

The demand for blood components is constantly increasing, while the exclusion criteria for donors are strengthened in order to reach maximal safety for donors and patients. To counterbalance reduced availability of volunteers, multicomponent collections (MCC) is an attractive approach to produce more than one component during a single apheresis procedure from one donor, such as packed red blood cells (PRBCs) and platelet concentrates (PCs). Further, the exposures of patients to a limited number of donors reduces the possibility of alloimmunization and transfusion-related diseases. We measured the quality of PRBCs and PCs obtained by MCC, using the MCS+ device with the LDPRBC program, Revision B, and compared them with the quality of manually collected PRBCs and PCs collected with the Revision C2 of the MCS+. We found higher pH levels and lower hemolysis assessed by means of fHb and K+ in the supernatant of PRBCs over the whole storage period of 42 days in MCC-derived PRBCs. The functional metabolism assessed by intracellular ATP was higher in PRBCs collected by MCC than in manually collected units. Furthermore, PCs obtained during MCC showed an increase in p-selectin expression on day 5 of storage compared to PCs collected with the Revision C2 of the MCS+. The p-selectin expression on MCC platelets was within the range of p-selectin expression found in PCs obtained by other apheresis devices. These results indicate less storage lesion in MCC-derived PRBCs compared to manually collected units and no compromise in the quality of MCC PCs obtained in the same apheresis procedure.     

The effect of storage on the expression of platelet membrane phosphatidylserine and the subsequent impacton the coagulant function of stored platelets

BACKGROUND: Platelet concentrates (PCs) derived from whole blood and stored under standard blood bank conditions undergo changes that are referred to as the platelet storage lesion. This study assesses the effect of PC preparation and storage on the distribution of phosphatidylserine (PS) in the platelet membrane and the effect that this distribution may have on the thrombogenic potential of stored PCs. STUDY DESIGN AND METHODS: Fresh platelets and PCs donated by healthy donors were obtained. PCs derived from platelet-rich plasma were studied on Day 1, Day 3, and Day 6 of storage under blood bank conditions. RESULTS: Platelet aggregation after exposure to the platelet agonists ADP and epinephrine singly declined progressively, but, when ADP and epinephrine in combination and collagen and thrombin in combination were used as agonists, the decline in platelet aggregation was less marked. PS expression as measured by Annexin V binding (mean and SD) was 2.02 +/- 0.93 percent in fresh platelet samples and increased to 5.39 +/- 4.2 percent on Day 1, 22. 1 +/- 7.1 percent on Day 3, and 39.5 +/- 12.1 percent on Day 6. Platelet prothrombinase activity (mean +/- SD) as measured by thrombin generation increased from 1.49 +/- 0.7 micro per mL in fresh platelet samples to 3.68 +/- 1.1 micro per mL in Day 1 platelets (p<0.001), 5.15 +/- 2.5 micro per mL in Day 3 platelets (p<0.001), and 4.65 +/- 2.48 micro per mL in Day 6 platelets (p<0. 001). CONCLUSION: These results show that PS expression increases after preparation of PCs from platelet-rich plasma and rises progressively during platelet storage under blood bank conditions. Furthermore, the greater PS expression is associated with increased platelet- dependent thrombin-generating capacity.     

Influence of storage time on activation of platelets collected with CS 3000 Plus and Cobe Spectra using platelet storage containers.

The aim of this study was to evaluate the in vitro activation in platelet suspensions collected with CS 3000 Plus and Cobe Spectra cell separators using platelet storage containers and the role of white blood cell (WBC) concentration of the suspension in this activation. Seventy-seven donors were subjected to automated platelet donations with 1 type of equipment (37 with Cobe Spectra and 40 with CS 3000 Plus). Blood samples were obtained immediately after separation and on the third day of storage at 22 degrees C in constant agitation. The WBC concentrations of these samples were studied before storage. Paraformaldehyde-fixed platelets were incubated with 2 murine monoclonal antibodies: CD42b and CD62. Murine monoclonal antibody immunoglobulin G was used as a negative isotypic control. Bound antibody was then quantitated by flow cytometry. On the third day of storage, a significant increase in CD62 expression rate was observed in platelet suspensions collected with both kinds of equipment. Mean expression rates for Cobe Spectra on Day 0 and Day 3 were 25.6 +/- 6.2% and 69.2 +/- 9.7%, respectively. Mean expression rates for CS 3000 Plus on Day 0 and Day 3 were 23.4 +/- 8.2% and 67.0 +/- 8.2%, respectively. The mean results for both devices were 22.8 +/- 4.56% for Day 0 and 68.7 +/- 13.2% for Day 3. There was no difference between CD42b mean fluorescence intensity on Days 0 and 3 for the 2 devices (p > 0.5). Mean WBC concentrations in the platelet suspensions for Cobe Spectra and CS 3000 Plus were 0.37 x 10(3)/microl and 0.42 x 10(3)/microl, respectively, and there was no relation between WBC concentration and increase in CD62 expression. Both kinds of equipment were found to be similar according to in vitro activation markers.     

The evaluation of microbial contamination in platelet concentrates prepared by two different methods.

The microbial contamination of platelet concentrates (PCs) prepared by two different methods both with a high risk of bacterial contamination during preparation and storage were evaluated. For apheresis platelets, the concentrates were obtained using the Haemonetics MCS 3P device. For the random method, platelets were obtained by two phase centrifugation, in the Heraeus Cryofuge 8500 I device using the Kansuk 3-way bags which permit storage for five days. 1620 plateletpheresis units prepared by apheresis, and 9838 units prepared by the random method, were included in the study. Of the 11,458 PCs studied. 32 (0.27%) were false positives and 24 (0.2%) were real positives. All of the positive results occurred in platelets prepared by the random method. C. xerosis and S. epidermidis, S. hominis, Alpha-hemolytic streptococci, all flora of the skin, were isolated in the contaminated concentrates. The risk of microbial contamination of PCs, prepared both by apheresis and from whole blood, continues at a low rate although the products were collected into specific bags following rules including appropriate disinfection of the skin, correct centrifugation collection time and optimal storage conditions including temperature and agitation. These results again emphasize the importance of: obeying phlebotomy rules and hand disinfection of the person who collects the blood as well as the need for careful skin decontamination of the donor, during donation.     

贮存血小板形态变化与凋亡因子磷脂酰丝氨酸表达的研究

目的探讨储存期间机采血小板形态变化、凋亡因子磷脂酰丝氨酸(phosphatidyl serine,PS)表达以及两者之间的关系,为确立血小板体外保存时限提供实验依据。方法应用May-Grunwald-Giemsa染色方法观察机采血小板储存0-8d时的形态变化,同时应用流式细胞术检测血小板的膜PS表达率。结果储存至第4天,血小板形态出现损伤,表现为形态学计分与新鲜血小板相比显著下降(t=2.341,P<0.05);随着储存时间延长,血小板形态学计分持续下降,至第7天,形态学计分下降31%。血小板凋亡因子PS表达,储存1d组与新鲜血小板0d组相比有明显升高(t=3.088,P<0.05);储存1-3d,PS表达无明显变化,储存至第4天,PS表达显著增加(t=2.1612,P<0.05);储存5-8d,PS表达持续增加,各组间有统计学差异(P均<0.05)。血小板形态学变化分值与膜PS的表达随储存时间延长呈负相关性(r=-0.9923,P<0.01)。结论储存血小板形态学变化分值与膜PS表达率高度负相关。     

In vitro platelet function of platelet concentrates prepared using three different apheresis devices determined by impedance and optical aggregometry

BACKGROUND: Testing the functional capacity of platelets (PLTs) from platelet concentrates (PC) is a main issue in transfusion medicine. Therefore, the aim was to study agonist-inducible PLT aggregation of PLTs obtained by three apheresis devices. A semiautomated impedance aggregometry-based whole-blood method (multiplate electrode PLT aggregometry [MEA]) was modified for the use of PLTs from PC and data were compared with light transmission aggregometry (LTA).
STUDY DESIGN AND METHODS: PLT function was determined in 135 PCs without reconstitution with red blood cells (RBCs), obtained by three devices: Amicus, Trima Accel collection system, and MCS+. PLT function was assessed by the Multiplate TRAP test, and the area under the curve (AUC) was quantified. TRAP-6–inducible maximal PLT aggregation (MA%) by LTA was used for analyses.
RESULTS: The AUC was significantly lower in the Amicus PLTs compared to the Trima and MCS+ PLTs (Amicus versus Trima, p = 0.007; Amicus versus MCS+, p < 0.001). The Amicus PLTs were significantly less responsive to TRAP-6–inducible PLT aggregation than Trima (p = 0.002) or MCS+ PLTs (p < 0.001) by LTA, and Trima PLTs responded significantly less than MCS+ PLTs (p = 0.001). There was only a weak correlation between MEA and LTA (r = 0.29, p = 0.019).
CONCLUSION: PLTs obtained by the Amicus system show significantly less aggregation response to thrombin receptor stimulation compared to those obtained with other cell separators, examined by MEA and LTA. Testing PLT function in PCs by the MEA is a simple and rapid method without the need of adding RBCs. However, LTA and MEA appear to measure different aspects of PLT function.     

Evaluation of the automated collection and extended storage of apheresis platelets in additive solution

BACKGROUND: Collecting apheresis platelets (PLTs) into additive solution has many potential benefits. The new Trima software (Version 6.0, CaridianBCT) allows automated addition of PLT additive solution (PAS) after collection, compared to Trima Version 5.1, which only collects PLTs into plasma. The aim of this study was to compare PLT quality during extended storage, after collection with the different Trima systems. STUDY DESIGN AND METHODS: Apheresis PLTs were collected using both Trima Accel apheresis systems. The test PLT units (n = 12) were collected using the new Trima Version 6.0 into PLT AS (PAS‐IIIM), while the control units (n = 8) were collected into autologous plasma using Trima Version 5.1. All units were stored for 9 days, and in vitro cell quality variables were evaluated during this time. RESULTS: PLTs collected in PAS‐IIIM maintained a stable pH between 7.2 and 7.4, whereas plasma‐stored apheresis units exhibited significantly increased acidity during storage, due to lactate accumulation and bicarbonate exhaustion. Plasma‐stored PLTs also demonstrated a more rapid consumption of glucose. However, there was little difference in PLT activation or cytokine secretion between PAS‐IIIM and control PLTs. CONCLUSION: These data indicate that apheresis PLT concentrates collected in PAS‐IIIM, using Trima Version 6.0 software, maintained acceptable PLT metabolic and cellular characteristics until Day 9 of storage.     

In vitro evaluation of COM.TEC apheresis platelet concentrates using a preparation set and pathogen inactivation over a storage period of five days

The aim of the present study was to evaluate in vitro data on platelets collected by apheresis, processed on a preparation set followed by photochemical treatment (PCT). Fifteen single-donor platelet concentrates (PCs) were collected by apheresis (COM.TEC blood cell separator, Fresenius, Bad Homburg, Germany). The platelets were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in approximately 37% plasma and 63% platelet additive solution InterSol. PCT was done by exposing the platelets to amotosalen HCl followed by illumination with ultraviolet light. Blood cell counts and in vitro PLT function were measured up to 5 days. An average of 3.44 +/- 0.28 x 10(11) platelets were collected in a product volume of 351 +/- 21 mL. Plasma removal resulted in a mean platelet loss of 7.8%. After PCT, a progressive decrease in platelet function was observed. LDH level rose through storage (171 +/- 81 U/L) to levels approximating LDH levels observed post-collection (180 +/- 103 U/L). There was a gradual decrease of the platelets to respond to hypotonic shock response from 90 +/- 9 % post-plasma reduction to 48 +/- 16% at day 5. All PLT units met the European requirements for leukoreduction and the pH limit of 6.8 up to day 5 post-collection. The new preparation set was capable of producing platelet units meeting the requirements for PCT. Despite differences observed in in vitro platelet function parameters, PLTs at storage day 5 fit the German and European guidelines.     

Analysis of the priming activity of lipids generated during routine storage of platelet concentrates

BACKGROUND: Compounds generated during the routine storage of platelet concentrates may have deleterious effects on the transfusion recipient. STUDY DESIGN and METHODS: Daily plasma samples from platelet concentrates, both apheresis platelets and those separated from whole blood, were obtained serially during routine storage. These plasma samples were assayed for their ability to prime the NADPH oxidase in isolated human neutrophils. Quantitative and qualitative analysis of the priming agents was completed by lipid extraction, high-pressure liquid chromatography separation, and gas chromatography/mass spectroscopy. RESULTS: Compounds were generated in both apheresis and whole-blood platelets that significantly primed the NADPH oxidase after 24 and 48 hours of storage, respectively. The priming activity was maximal by component outdate: 2.6-fold that of the buffer-treated control neutrophils (apheresis) and 3.9-fold that of the buffer-treated control neutrophils (whole blood). These agents were generated by cellular constituents, as stored plasma did not demonstrate such priming activity. Inhibition of this priming activity by WEB 2170, a specific platelet-activating factor receptor antagonist, suggested that the observed priming involved the platelet-activating factor receptor. A portion of the priming activity from platelet concentrates was organically extractable: 69 percent of that from apheresis platelets and 46 percent of that from whole-blood platelets. Further purification of the lipid's priming activity by normal-phase high-pressure liquid chromatography demonstrated a single peak of priming activity at the retention time of lysophosphatidylcholines. Because 46 percent of the priming activity from whole-blood platelets was chloroform insoluble and because it has been reported that interleukin 8 is generated during routine storage of whole-blood platelets, the effects of interleukin 8 on the NADPH oxidase were examined. Recombinant monocyte interleukin 8 rapidly primed the oxidase but was not inhibited by WEB 2170. CONCLUSION: Lipids were generated during the routine storage of platelet concentrates that prime the NADPH oxidase, and they may play a role in the severe complications of transfusion therapy. Other non- lipid compounds, such as interleukin 8, that are generated in whole- blood platelets may also contribute to the observed priming activity of plasma.     

L-carnitine improves pH and decreases surface phosphatidylserine expression in extended stored apheresis platelets

Extension of the storage period of apheresis platelets to seven or ten days may be possible with the implementation of screening for bacteria. This, however, may impair platelet quality, and additive compounds that improve storage parameters would be desirable. Apheresis platelets were harvested using the Cobe LRS device. Part of the product was aliquoted into two CLX bags, 60 ml into each, on day 0. L-carnitine (LC) to a final concentration of 5 mM was added to one container and saline to the other. pH, morphology score, and surface expression of phosphatidylserine were measured on day 1, and, in addition, hypotonic shock response (HSR) and the extent of shape change (ESC) on days 5, 10, and 13. Differences between test and controls were analyzed using paired t-tests. The addition of LC improved pH by day 5, but was more evident by days 10 and 13. By day 10, significant differences (<0.01) were observed in pH (6.54 +/- 0.3 vs. 6.75 +/- 0.3), lactate (176 +/- 31 vs. 150 +/- 24 mg %), morphology score (213 +/- 27 vs. 229 +/- 35) and ESC (7 +/- 6 vs. 11 +/- 6). Percent surface phosphatidylserine expression was less in the LC treated platelets (16 +/- 7 vs. 12 +/- 4, P<0.03). Much of the benefit observed was attributable to improved parameters in some donors. LC improves the quality of extended stored apheresis platelets.     

Binding of activated platelets to WBCs in vivo after transfusion

BACKGROUND: During preparation and storage of apheresis concentrates, platelets are being activated. One of the alterations that occur during this process is an increased expression of P-selectin (CD62p) on the cytoplasmic surface of platelets. This neoepitope represents a ligand for the binding of platelets to WBCs. It has been suggested that the activation of platelets is associated with the sequestration of platelets after transfusion. In this in vivo study, the binding of platelets to WBCs was analyzed following transfusion of platelet concentrates (PCs). STUDY DESIGN AND METHODS: Double apheresis concentrates were prepared with two different cell separators. One of the split products was stored for 1 to 2 days and the other one for 3 to 5 days. Flow cytometry was applied to analyze the degree of platelet activation in vitro, and also to measure the extent of platelet binding to WBC subclasses in vivo after transfusion into patients. RESULTS: The results of this study show that platelet activation occurs during apheresis and storage of PCs. After transfusion of the PCs, no significant binding of platelets to T or B-cells could be detected. However, a significant binding of platelets to monocytes and neutrophil granulocytes occurs. While in Baxter PCs stored for 1-2 days the amount of platelet-leukocyte aggregates in vivo was higher compared to COBE PCs, no such difference could be detected anymore for the PCs stored for 3-5 days. CONCLUSION: This study demonstrates that binding of activated platelets occurs to monocytes and neutrophil granulocytes but not to T- and B-cells in the circulation after transfusion. In addition, the interaction of platelets and WBCs is dependent on the degree of P-selectin expression. Platelets showing a higher degree of activation adhere to WBCs to a higher degree than nonactivated platelets.     

Evaluation of Cobe Trima for the collection of blood components with particular reference to the in vitro characteristics of the red cell and platelet concentrates and the clinical responses to transfusion

This study evaluated Cobe Trima for donor and operational acceptability, the quality and storage stability of the blood components collected, and the clinical responses to transfusion. The study was carried out in 2 phases; phase 1 assessed the efficiency of red cells and platelet collection, and the characteristics of the components collected before and after storage. Phase 2 was an evaluation of operational issues and the in vitro characteristics of the red cells and platelet concentrates at the time of transfusion in respect to their cellular content, and leucocyte (interleukin IL-6 and IL-8) and platelet-derived (Rantes) cytokine levels. Cytokine levels were also measured in the donors before and after the collection procedure and in patients both before and after transfusion. The clinical responses to a small number of transfusions were assessed. The Cobe Trima was found to be straightforward to use by the operators, although additional operator training was required to manage occasional uncertainty with alarm messages. It was acceptable to the donors except for the occurrence of citrate reactions in 3/6 donors in phase 1; this problem persisted in phase 2 (6/15 donors), and needs to be addressed in the future. All blood components met UK product specifications apart from 2 platelet concentrates, 2 red cell concentrates, and one unit of FFP; the red cell and platelet concentrates had good storage characteristics. The 2 procedures, which resulted in low platelet yields, were due to occlusion of the plasma line; the method for installation of the harness has been subsequently modified to prevent this. 2 red cell concentrates showed haemolysis; the reason for this was not established. The Factor VIII level was satisfactory in plasma and the cellular content was low. The responses to 12 platelet transfusions were expected as in a group of haematology patients, and no immediate adverse effects were reported with any of the transfusions. Leucocyte-associated (IL-8 and IL-6) and platelet-associated (Rantes) cytokine levels were not elevated in donor samples taken before or after the collection procedure, or in the red cell and platelet concentrates at the time of issue. Pre- and post-transfusion IL-8 levels were raised in one patient with non-immune platelet refractoriness, and normal in 2 patients with excellent or almost satisfactory responses to platelet transfusions raising the question as whether IL-8 could be used as a laboratory marker for non-immune platelet refractoriness due to infection.     

Apoptotic activity in stored human platelets

BACKGROUND: Platelets possess some of the machinery required for apoptotic cell death. However, disruption of mitochondria function, implicated in several models of cell death, has not been extensively studied in platelets. Mitochondrial viability and several other measures of apoptotic death in stored and experimentally stressed platelets were evaluated. MATERIALS AND METHODS: Platelet mitochondrial transmembrane potentials (Deltapsim) were studied by staining platelets with JC-1, a dye that fluoresces at different wavelengths based on the state of mitochondrial polarization. Annexin V binding, a measure of phosphatidylserine (PS) exposure, and CD62P expression, an indicator of platelet activation, were determined by flow cytometry. Caspase-3 activity was measured with an enzyme assay and by Western blotting. Experimental platelet stressors included storage for 7 days, azide exposure, calcium ionophore stimulation, and plasma deprivation. RESULTS: As measured by flow cytometry, Deltapsim values were similar in freshly drawn platelets and in platelet concentrates stored for up to 7 days. However, compared to fresh platelets, stored platelet concentrates had significantly increased PS exposure (3.1 vs. 5.1%, p = 0.015), CD62P expression (6.5 vs. 13.5%, p = 0.0067), and caspase-3 activity. Azide exposure, which decreased ATP release 20 to 30 percent, did not affect the Deltapsim. Stressed platelets exhibited higher degrees of mitochondrial depolarization in response to calcium ionophore stimulation than platelets that were not stressed. Plasma deprivation also resulted in significant alterations in Deltapsim, PS exposure, and CD62P expression. CONCLUSIONS: Platelet mitochondria maintain Deltapsim when stored for up to 7 days under standard blood bank storage conditions. Therefore, changes in platelet mitochondria Deltapsim do not correlate with downstream markers of apoptotic death such as caspase activation and PS exposure.     

In vivo recovery and survival of apheresis and whole blood-derived platelets: a paired comparison in healthy volunteers

BACKGROUND: Methods of platelet preparation may alter the recovery and survival characteristics of platelets following transfusion. As suggested by a recent clinical trial, platelet recovery may be better preserved with apheresis platelet preparations than with platelets prepared from whole blood by the platelet-rich plasma (PRP) method. STUDY DESIGN AND METHODS: In vivo platelet recovery and survival of autologous leukoreduced (LR) apheresis platelets and autologous filter-LR PRP platelets were compared in 22 healthy volunteers using a paired crossover design. On the same day, each participant gave one apheresis platelet donation and one whole blood donation from which platelets were recovered from the PRP. The sequence of donations was randomly assigned for each participant. Following 5 days of storage and bacterial screening, a sample from each platelet product was labeled with either (51)chromium or (111)indium (randomly assigned) and both samples were simultaneously re-infused into the original donor. Recovery and gamma-function platelet survival were calculated for each platelet product using the multiple hit mathematical model. RESULTS: Five day stored LR-apheresis platelets had 18.8 percent better recovery, and 32.9 percent longer gamma-survival than filter-LR PRP platelets. Stored apheresis platelets had lower p-selectin expression and higher morphology scores than stored PRP platelets. CONCLUSIONS: Filter-LR PRP platelet preparation appears to adversely affect platelet recovery and survival characteristics. The reasons for this effect are not clear. These results may not apply to all apheresis and PRP methods of platelet preparation.