In vitro function of buffy coat-derived platelet concentrates stored for 9 days in CompoSol, PASII or 100% plasma in three different storage bags

BACKGROUND AND OBJECTIVES: The aim of the study was to compare the in vitro quality of buffy coat-derived platelet concentrates (PC) during extended storage in plasma or additive solution in three different storage bags. MATERIALS AND METHODS: A pooled and split design was chosen so that identical PCs were produced in either 100% plasma, 70% PASII : 30% plasma or 70% CompoSol : 30% plasma (n = 6 each). This was repeated for three different manufacturers' platelet storage bags (Fresenius, Baxter and Pall). PCs were sampled on days 1, 5, 7 and 9 of storage and tested in vitro using a variety of tests of platelet function. For each bag type, storage in PASII or Composol was compared with plasma (data taken across the entire storage period), and differences occurring with time were analysed for all storage media. RESULTS: The pH of all PCs was > 6.8 at day 9 of storage. In vitro platelet function, as assessed by markers of platelet activation and metabolism, of PCs stored in CompoSol appeared to be similar to that of PCs stored in plasma over 9 days of storage. In contrast, PCs stored in PASII tended to have significantly higher levels of platelet activation (almost a twofold increase in % platelets positive for CD62P by day 5) and lower hypotonic shock response (approximately 40%, by day 7) compared to either PCs stored in 100% plasma or 70% CompoSol. The magnitude of the differences observed between platelet storage media appeared to be dependent on the type of platelet storage bag with the highest degree of platelet activation and lowest hypotonic shock response values being observed in Fresenius bags in combination with PASII. CONCLUSIONS: The maintenance of platelet function in vitro during extended storage of PCs in platelet additive solutions is dependent on the combination of type of additive solution and type of platelet storage bag. For all bag types studied, storage in PASII resulted in poorer platelet function in vitro.     

Agonist-inducible platelet activation in chronic idiopathic autoimmune thrombocytopenia

OBJECTIVE: There are only few studies on agonist-inducible platelet activation in chronic idiopathic autoimmune thrombocytopenia (cAITP). MATERIALS AND METHODS: We compared agonist (TRAP-6, ADP, Arachidonic acid, Epinephrine, and Ristocetin) -inducible P-selectin expression and PAC-1 binding in 40 patients with cAITP (f/m ratio 23/17) with those in 20 healthy controls. Results were correlated with platelet counts, detectable platelet antibodies, and reticulated platelets. RESULTS: The in vivo activation of platelets determined the in vitro inducible response to agonists. The stronger the in vivo activation the less the number of platelets responding to agonists, as illustrated by the inverse correlation of P-selectin expression ex vivo and the relative increase after the exogenous addition of agonists. The agonist-inducible platelet activation was not associated with the presence of detectable platelet antibodies to GPIb/IX or GPIIb/IIIa. Agonist-inducible platelet activation was also not correlated with counts of reticulated platelets. CONCLUSION: Agonist-inducible activation of platelets in cAITP is affected mainly by their in vivo activation.     

In vitro function of platelet concentrates prepared after filtration of whole blood or buffy coat pools

BACKGROUND/METHOD: Data on the quality of platelet concentrates (PC) produced by the buffy coat method and stored beyond 5 days in plasma are limited. We therefore evaluated the quality of PCs prepared by leucocyte depletion of whole blood (Terumo WBSP, n = 10) or a buffy coat pool (Pall Autostop, n = 10), and stored for 7 days in plasma by assessing platelet parameters and markers of platelet activation. RESULTS: In both types of PC, levels of glucose decreased during storage but were not totally depleted (> 11 mM on day 7). In contrast, lactate levels increased on storage and was consistently < 20 mM throughout, with pH maintained at > 6.8 in all units. Hypotonic shock response scores were > 47% in all units at day 7. On day 1, markers of platelet activation were significantly higher in WBSP PC, but by day 7 were similar for percentage CD63+ and CD62P + (40%) with levels of platelet microparticles and annexin V binding two-fold higher in WBSP. The expression of CD61 did not alter during storage and the percentage of platelets expressing CD42b was > 88% in all units on day 7. RANTES (Regulated on activation, normal, T-cell expressed and secreted) and TGFbeta released from platelets by day 7 was < 800 ng/ml and 90 ng/ml, respectively. C3a(desarg) increased throughout storage in both types of PC, but without a commensurate increase in the terminal complex SC5b-9 or activation of factor XII. CONCLUSION: Our data indicates that the in vitro characteristics of PCs prepared using these methods is maintained over storage for 7 days in plasma and is not associated with significant deterioration of platelet function. ONE SENTENCE SUMMARY: In vitro function of platelet concentrates prepared by either filtration of whole blood, or pooled buffy coats.     

Storage of platelets in additive solutions: the effects of magnesium and potassium on the release of RANTES, beta-thromboglobulin, platelet factor 4 and interleukin-7, during storage

BACKGROUND AND OBJECTIVES: Several studies have suggested that the accumulation of cytokines during storage of platelet concentrates may mediate non-haemolytic transfusion reactions. Prestorage leucodepletion can prevent the release of cytokines from white blood cells during storage, but not the release of platelet-derived cytokines. Therefore, we investigated whether the addition of magnesium and potassium to platelets stored in a platelet additive solution (PAS) would affect the generation of cytokines during platelet storage. MATERIALS AND METHODS: Platelets were prepared from buffy coats using different suspension media: plasma; 70% PAS-III + 30% plasma; 70% PAS-III supplemented with magnesium and potassium +30% plasma; and 80% PAS-III supplemented with magnesium and potassium +20% plasma. The levels of certain cytokines--regulated on activation, normal, T-cell expressed, and secreted (RANTES), beta-thromboglobulin (beta-TG), platelet factor 4 (PF4) and interleukin-7 (IL-7)--were measured by enzyme-linked immunosorbent assay (ELISA) on days 1, 5 and 7. RESULTS: The concentrations of RANTES, beta-TG, PF4 and IL-7 increased, during storage, in all units. The increase was significantly greater in units stored in 70% PAS-III +30% plasma than in the other three suspension media. The storage of platelets in 70% PAS-III supplemented with magnesium and potassium +30% plasma significantly reduced the concentrations of platelet derived-cytokines during storage, as compared to platelets stored in 70% PAS-III + 30% plasma alone. CONCLUSIONS: The concentrations of platelet-derived cytokines increased, to a significantly greater extent, when platelets were stored in PAS-III than in plasma. However, when magnesium and potassium were added to PAS-III, the concentrations of platelet-derived cytokines obtained during storage were about the same as those produced by platelets stored in plasma.     

Effect of heparin on platelet count and platelet aggregation

The in vitro effect of heparin on platelet aggregation was studied in three groups: in 26 subjects recently treated with heparin, in 18 subjects on maintenance hemodialysis, and in 20 normal controls. With the aid of Technicon H6000, platelet counts and platelet aggregations were compared in whole blood samples collected in ethylenediaminetetraacetic acid (EDTA) and in heparinized tubes. Although there was no significant difference between platelet count of heparinized and EDTA blood in the control group, the dialysis group and the group recently treated with heparin showed significantly lower platelet counts and more platelet aggregation in heparinized tubes than in EDTA tubes. We speculate that the majority of subjects exposed to heparin develop an antibody or a proaggregator which can aggregate or agglutinate platelets in the presence of heparin and causes destruction of platelets; but only in a small percentage of subjects receiving heparin is this reaction severe enough to cause thrombocytopenia.     

Improving platelet transfusion safety: biomedical and technical considerations

Platelet concentrates account for near 10% of all labile blood components but are responsible for more than 25% of the reported adverse events. Besides factors related to patients themselves, who may be particularly at risk of side effects because of their underlying illness, there are aspects of platelet collection and storage that predispose to adverse events. Platelets for transfusion are strongly activated by collection through disposal equipment, which can stress the cells, and by preservation at 22 °C with rotation or rocking, which likewise leads to platelet activation, perhaps more so than storage at 4 °C. Lastly, platelets constitutively possess a very large number of bioactive components that may elicit pro-inflammatory reactions when infused into a patient. This review aims to describe approaches that may be crucial to minimising side effects while optimising safety and quality. We suggest that platelet transfusion is complex, in part because of the complexity of the “material” itself: platelets are highly versatile cells and the transfusion process adds a myriad of variables that present many challenges for preserving basal platelet function and preventing dysfunctional activation of the platelets. The review also presents information showing - after years of exhaustive haemovigilance - that whole blood buffy coat pooled platelet components are extremely safe compared to the gold standard (i.e. apheresis platelet components), both in terms of acquired infections and of immunological/inflammatory hazards.     

Sterilization method of platelet storage containers affects in vitro parameters

BACKGROUND AND AIM: Four butyryl trihexyl phthalate plasticized polyvinyl chloride (BTHC-PVC) containers were compared for storage of leukoreduced platelet concentrates (LR-PC): three ethylene oxide (EtO) sterilized (Gambro, Haemonetics, Fresenius), and one steam-sterilized (Fresenius). METHODS: LR-PCs were made from 5 buffy coats and 300 ml Composol additive solution, and leukoreduced by filtration. Four LR-PCs were pooled and subsequently divided over the 4 BTHC-PVC bags to prevent donor-dependent differences, and sampled for in vitro analysis on day 1, 2, 5, 7 and 9. RESULTS: The pH values on day 9 were (mean +/- SD, n = 10): 7.12 +/- 0.03 (Gambro), 7.12 +/- 0.04 (Haemonetics), and 7.07 +/- 0.09 (Fresenius, EtO-sterilized) (not significantly different), vs. 6.91 +/- 0.12 (Fresenius, steam-sterilized; P < 0.001 vs. all EtO-sterilized bags). LR-PCs stored in the steam-sterilized bag exhibited significantly higher glucose consumption and lactate production (P < 0.001 vs. all EtO-sterilized bags). CONCLUSION: All BTHC-PVC containers allow storage of LR-PCs for up to 9 days with good in vitro parameters. However, the method of sterilization affects the storage conditions of the LR-PCs in these bags.     

Use of the DiaMed Impact R to test platelet function in stored platelet concentrates

BACKGROUND AND OBJECTIVES: The DiaMed Impact R tests platelet function under close to physiological flow conditions using cone and plate technology. An image analyser quantifies the adhered platelets and results are expressed as percentage of well surface covered by aggregates (SC %) as an index of adhesion and average size of the aggregates (AS microm(2)) as an index of aggregation. The machine is designed to use whole blood and the aim of this study was to determine if it could be used to assess platelet function in platelet concentrates (PC). MATERIAL AND METHODS: Platelet concentrates were mixed with various ratios of platelet-poor plasma (PPP) or ABO-compatible routine leucoreduced red cells in saline-adenine-glucose-mannitol. The effects of platelet counts, haematocrit, shear rate and time of activation upon SC and AS were evaluated to identify optimized assay conditions. Routine PCs were then tested on Days 2, 5 and 7. Samples were also stored at 4 or 37 degrees C for 1 to 2 h before assay to see if function was altered. RESULTS: Platelet concentrate in PPP resulted in no detectable platelet adhesion. However, addition of red cells to PC resulted in measurable platelet adhesion and aggregation. Optimal conditions were identified as shear rate of 1800 per second, 4-min activation, platelet count between 250 and 400 x 10(6) per ml, haematocrit between 30 and 40%. When stored PCs were tested under these conditions we observed median values of 8.2% for SC and 32.7 microm(2) for AS at 2 days, which reduced to 6.9% and 25.0 microm(2), respectively, after 5-day storage and 6.8% and 21.0 microm(2) after 7 days. CONCLUSION: We were able to reconstitute PCs by adding red cells and identified conditions to allow platelet adhesion and aggregation functions of PCs to be measurable in the DiaMed Impact R. Platelet functions of adhesion and aggregation were shown to decrease during storage but improved after a 1-h treatment at 37 degrees C.     

Correlation between the extent of platelet activation in platelet concentrates and in vitro and in vivo parameters

BACKGROUND AND OBJECTIVES: Platelet activation, which is necessary to stop bleeding, also occurs in vitro during the storage of platelet concentrates (PCs). However, it is unknown whether in vitro-activated platelets are able to reduce blood loss in the patient. We studied correlations between platelet activation in PCs and in vitro parameters (pH, platelet count, swirling effect, storage time). In addition, we studied the correlation between platelet activation and in vivo parameters [the volume of thorax drain fluid as a measure of blood loss, platelet count, international normalized ratio (INR), and activated partial thrombin time (APTT)] in a clinical pilot study. MATERIALS AND METHODS: White blood cell-reduced PCs prepared from five buffy coats and one plasma unit (n = 55; storage time: median, 5 days; range, 2-12 days) were sampled. Platelet activation (CD62p, CD63, CD42b), as measured by flow cytometry, pH, platelet count, swirling effect and storage time, was determined. For the in vivo pilot study, PCs (n = 21) stored for 2-7 days were also checked for the above parameters and transfused into patients (n = 21) immediately after coronary artery bypass graft surgery. The volume of thorax drain fluid was measured for up to 12 h after surgery, and the platelet count, INR and APTT were measured < 1 h and 16-24 h postsurgery. RESULTS: A good correlation (r2 > 0.5) was observed between CD62p and CD63, between CD62p and CD42b, between CD62p or CD63 and pH and between CD62p or CD63 and swirling effect. Also, a significant increase in platelet activation was observed for PCs stored for > or = 8 days (mean +/- standard deviation: CD62p, 41.6 +/- 30.7; CD63, 23.8 +/- 18.6), compared to PCs stored for 2-7 days (mean +/- standard deviation: CD62p, 12.3 +/- 4.8; CD63, 10.4 +/- 3.6). No correlation (r2 < or = 0.1) was observed between platelet activation and the in vivo parameters. CONCLUSIONS: Although a correlation between platelet activation and in vitro parameters was observed, no correlation was found between platelet activation and in vivo parameters. Possible explanations for this are a too low variance in platelet activation in transfused PCs, and too small a number of patients.     

In vitro and in vivo effects of potassium and magnesium on storage up to 7 days of apheresis platelet concentrates in platelet additive solution

BACKGROUND AND OBJECTIVE: Prolonged storage of platelets up to 7 days provides improved availability, logistical management and decreased wastage. Beside methods of bacterial detection, addition of magnesium and potassium to the platelet storage solution (SSP+) may further improve the quality of platelets with extended storage. MATERIALS AND METHODS: Apheresis platelets from 10 donors were divided and stored in two different platelet additive solutions (PAS) (Intersol and SSP+) for a paired comparison. A variety of in vitro platelet function and metabolic assays were performed both on day 1 and after 7 days of storage. For in vivo study, platelets were labelled with either (111)Indium or (51)Chromium after 7 days of storage and were injected into the corresponding donor. Serial blood samples were drawn for recovery and survival measurements. RESULTS: In vitro parameters for SSP+ showed significantly reduced glycolysis (lower glucose consumption and decreased production of lactate), a higher hypotonic shock response (HSR) and the extent of shape change reactivity and a lower degree of platelet activation by means of RANTES (regulated on activation, normal, T cell-expressed, and secreted), CD62p and CD63 expression. Platelet recovery on day 7 was higher for Intersol as compared to SSP+, 65 +/- 11 vs. 53 +/- 13% (P = 0.023), and survival showed no difference 4.2 +/- 1.9 vs. 3.6 +/- 1.4 days. CONCLUSION: In vitro characteristics of platelets stored in PAS with addition of potassium and magnesium indicated higher quality, but this could not be verified by the in vivo parameters by means of recovery and survival.     

Extended storage of platelets in SSP platelet additive solution

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate the effects of extended storage of pooled random platelets in SSP+ additive solution (MacoPharma). MATERIALS AND METHODS: Eight buffy coat-derived, pooled, leucoreduced platelet concentrates were prepared in 75% SSP+, 25% plasma using Fresenius/NPBI Composelect thrombocyte polishing filter (TPF) systems. Platelet concentrates were stored for 19 days in polyolefin storage bags and samples for in vitro analysis were taken at various time-points during storage. RESULTS: Platelet yields were lower than seen routinely when platelets are prepared in 100% plasma. The in vitro quality of the platelets stored in SSP+ was maintained until day 9. Glucose was depleted by day 12 and this was accompanied by a rapid fall in pCO2, a rise in pO2 and a cessation of lactate production. ATP and bicarbonate concentrations fell, the platelets began to swell and the ability to swirl decreased. Soluble P-selectin, glycocalicin, and regulated on activation, normal, T-cell expressed, and secreted (RANTES) concentrations increased, as did P-selectin expression. Loss of platelets and an increase in lacate hydrogenase concentration indicated that lysis had occurred. However, the pH remained between 6.4 and 7.4. CONCLUSIONS: The results suggest that SSP+ could be used for platelet storage for up to 9 days. However, the preparation of platelets in the additive requires some optimization. In vivo studies are required to confirm these in vitro results.     

Stored red‐blood‐cells inhibit platelet function under physiologic flow

Background and Objectives The DiaMed Impact R tests platelet function under close to physiological flow conditions. The machine is designed to use whole blood but by adding back compatible red cells, it can be used to study stored platelet concentrates. To date, red cells ≤14 days old have been used. In this study, the effect on the assay of using red cells stored for up to 60 days was examined. Material and Methods This study looked at buffy coat‐derived platelet concentrates on day 2 of storage along with various stored red‐blood‐cells (RBC). To determine whether the age of the RBC is a factor in supporting adhesion and aggregation, platelets were assayed with either RBC stored between 2 and 60 days or with separated ‘young’ and ‘old’ red cell populations obtained using a centrifugation method and confirmed by percoll gradient analysis. Results A statistically significant difference was observed between red‐blood‐cells stored for ≤20 days compared with those which have been stored for 21–60 days in respect of their ability to support platelet adhesion (SC) and aggregation (AS) (P < 0·01). Separating red cells by centrifugation into top (young population) and bottom (old population) showed that the effect of storage was much greater than was any difference between young and old at the individual time‐points e.g. ‘young’ red cells from stored units were poorer at supporting platelet adhesion and aggregation than ‘young’ red cells from fresh units. Conclusion Results suggest that the red cells should be stored for less than 21 days when using this assay. This assay may also allow assessment of red cell functionality.     

Acoustophoretic purification of platelets: feasibility and impact on platelet activation and function

Purity, limited platelet activation, and preservation of platelet function are important stakes of preparation of platelet concentrates (PC) for clinical use. In fact, contaminating red blood cells and leukocytes, as well as activated and/or poorly functional platelets in PC, represents a risk of poor efficiency and adverse side effects during platelet transfusion. Therefore, optimization of preparation and storage of PC is still an active field of research. Shear-induced platelet activation is an unwanted side effect of the hard-spin (up to 5000g) step of centrifugation-based methods currently used in blood banks to prepare PC from whole blood samples. Here, we evaluated the effectiveness of an acoustic-based fractionation device for the isolation of human platelets from whole blood bags. The purity, activation status, and functionality of platelets isolated by acoustopheresis were compared with those of platelets isolated using a reference protocol known to produce limited platelet activation and consisting of two consecutive soft-spin centrifugations (120g and 1200g). Platelet concentration and purity were determined using an automated hematology analyzer. Platelet activation status and platelet reactivity to collagen and thrombin were assessed in flow cytometry by measurement of surface expression of P-selectin and activated integrin αIIbβ3. The ability of isolated platelets to incorporate into a thrombus when transfused to NOD/SCID mice was investigated by intravital microscopy using the ferric chloride-induced thrombosis model. Blood fractionation by acoustophoresis led to the elimination of more than 80% of red blood cells and leukocytes from the platelet fraction, whose mean purity was of 92.8 ± 12.8%. The activation status and reactivity to collagen and thrombin of acoustophoresis-isolated platelets were similar to those of platelets isolated by soft-spin centrifugation. Finally, acoustophoresis-isolated platelets were tethered, adhered to the vessel wall, and incorporated into a growing thrombus following ferric chloride-induced vascular injury. Together, our results indicate that acoustophoresis is a suitable method for the isolation of human platelets with minimal platelet activation and preservation of platelet function.     

Pathogen reduction of buffy coat platelet concentrates using riboflavin and light: comparisons with pathogen-reduction technology-treated apheresis platelet products

BACKGROUND AND OBJECTIVES: A pathogen-reduction technology (PRT) system using riboflavin and light has been developed for the treatment of platelet concentrates (PC) obtained by either buffy coat preparation (BCPC) or apheresis procedures (APPC). The aim of this study was to evaluate the effects of the treatment process on in vitro cell quality and on riboflavin conversion in PC. MATERIALS AND METHODS: BCPC were prepared with the Compomat G4 from whole blood which had been stored overnight after collection. APPC were obtained using the TRIMA apheresis procedure. Both PC products had been stored for 18-24 h prior to PRT treatment. BCPC and APPC were treated with PRT on day 2 and day 1 of shelf-life, respectively. The treated PCs were then maintained for an additional 5 days after the PRT treatment. A panel of cell quality assays and high-performance liquid chromatography (HPLC) analysis were performed. RESULTS: Cell counts and plasma lactate dehydrogenase (LDH) levels during storage indicated that PRT did not induce significant cell lysis. Acceleration of a decrease in glucose and an increase in lactate was observed for treated PCs, but no significant differences were observed between treated BCPC and APPC. The pH of treated samples remained above 7.0, although was lower than that of the control. Platelet morphology of BCPC and APPC was well preserved. P-selectin expression indicated significant platelet activation when compared with control PC (BCPC on day 6: 39% vs. 12%; APPC on day 5: 35% vs. 18%). Both P-selectin expression and microparticle formation were not significantly different between treated BCPC and APPC during storage. The JC-1 assay also displayed no loss of mitochondria integrity during the storage of treated products. Approximately 20% of riboflavin converted into photoproducts, including lumichrome. CONCLUSIONS: PRT treatment had an effect on the development of the normal platelet storage lesion at a level which seems tolerable for clinical usage.     

Evaluation of platelet function in thrombocytopenia

Whole blood aggregometry is a functional assay for determination of platelet function. Until now, whole blood aggregometry has not been considered feasible at low platelet counts. Hence, the objectives of the present study were to explore platelet function in thrombocytopenia using a novel index of impedance aggregometry adjusted for platelet count and evaluate the association to platelet function assessed by flow cytometry. Hirudin anticoagulated blood was collected from 20 healthy volunteers, 20 patients with primary immune thrombocytopenia (ITP), and 17 hematological cancer patients. Platelet function was analyzed by impedance aggregometry and by flow cytometry. Collagen, adenosine diphosphate, thrombin receptor agonist peptide-6, and ristocetin were used as agonists for both analyses. Thrombocytopenia in healthy whole blood was induced in vitro employing a recently published method. Platelet aggregation of thrombocytopenic patients was evaluated relative to the aggregation of healthy volunteers at the same platelet count. In flow cytometry, platelet function was described as expression of the platelet surface glycoproteins: bound fibrinogen, CD63, and P-selectin. Similar platelet counts were obtained in the patient groups (p = 0.69) (range: 13–129 × 10⁹/l). Aggregation adjusted for platelet count was significantly increased in ITP patients compared to healthy platelets across all agonists. The platelet aggregation was high in the 95% prediction interval, with 18 ITP patients above the prediction interval in at least two agonists. In contrast, the platelet aggregation was low in the prediction interval in cancer patients, and three cancer patients with platelet aggregation below the prediction interval in at least one agonist. ITP patients displayed increased expression of bound fibrinogen and CD63 following activation, compared with particularly cancer patients, but also compared with healthy platelets. This study demonstrated the feasibility of a novel approach to perform platelet function analyses in thrombocytopenia using impedance aggregometry adjusted for platelet count.     

5-day storage of human platelet concentrates in 30 ml of plasma or artificial medium

Optimal conditions for the storage of platelet concentrates were studied by changing 5 environmental parameters: bag composition (PL146 vs. PL732), volume of plasma (60 vs. 30 ml), anticoagulant (CPDA-1 vs. heparin), nutrient (glucose vs. fructose) and medium (plasma vs. artificial medium). A full bilevel factorial study was conducted to evaluate each variable alone and in combination with the other variables for their effects on platelet aggregation and release in response to single and pairs of stimuli. Serotonin uptake, pCO2, platelet count, lactate, glucose, pO2, pH and white blood cell concentration were also measured after 3 and 5 days of storage. Platelets that were stored in PL146 bags had reduced responses to stimulation by 3 days and markedly impaired responses after 5 days relative to platelets that were stored in PL732 bags. There was a large drop in pH and platelet responsiveness when platelets were stored in a volume of 30 ml in PL146 bags; these were not found when platelets were stored in 30 ml in PL732 bags. Replacing plasma with an artificial medium or adding fructose or heparin and calcium to plasma yielded platelets that were equally functional as routine controls in CPD-A1 plasma. It was concluded that replacement of plasma with 60 ml of artificial medium or a reduction of plasma volume with storage in PL732 bags are two possible mechanisms of obtaining more plasma from blood donations without compromising maximum platelet storage life.     

In vitro platelet adhesion to vascular subendothelium: studies in patients with polycythaemia vera

In this work we studied platelet adhesion to subendothelial surfaces in 10 patients with polycythaemia vera and 10 healthy volunteers at 40% Hct (corresponding to the mean value of our control group) and 55% Hct (a value roughly corresponding to the mean Hct in polycythaemic patients). Platelet concentration was kept constant at 2.0-2.5 X 10(11)/l. The results indicate that there was a statistically significant increase in adhesion both in controls and in patients with Hct varying from 40% to 55%. The contribution of the higher Hct in promoting platelet adhesion was comparable in the two groups. When red blood cells (RBC) from the patients were tested with platelets from healthy volunteers in cross-over experiments, they promoted adhesion in the same way as control RBC. Similarly, when patients' platelets were mixed with control RBC, adhesion was the same as control platelets. These data indicate that platelet and RBC contribution to this parameter are not significantly modified in this group of polycythaemic patients, provided that platelet and RBC values are adjusted to control range.     

Six years' experience of using the BacT/ALERT system to screen all platelet concentrates, and additional testing of outdated platelet concentrates to estimate the frequency of false-negative results

BACKGROUND AND OBJECTIVES: Approximately 1 in every 2000 units of platelets is contaminated with bacteria. The BacT/ALERT automated blood culture system can be used to screen platelet concentrates (PCs) for bacterial contamination. MATERIALS AND METHODS: Data were collected from May 1998 until May 2004. The number of PCs tested during this period was 36 896, most of which were produced from pools of four buffy-coats. On the day following blood collection or platelet apheresis, a 5-10 ml sample of the PC was aseptically transferred to a BacT/ALERT culture bottle for detection of aerobic bacteria. The sample was monitored for bacterial growth during the entire storage period of the PC (6.5 days). When a positive signal was generated, the culture bottle, the PC and the erythrocyte concentrates were tested for bacterial growth. In order to determine the frequency of false-negative BacT/ALERT signals, 1061 outdated PCs were tested during the period from May 2002 to May 2004. RESULTS: Eighty-eight positive signals were detected by the BacT/ALERT system, of which 12 were interpreted as truly positive. Fourteen signals were interpreted as truly false positive. Thirty-three signals were interpreted to be probably false positive. Two of 1061 outdated units tested positive, and Bacillus spp. and Staphylococcus epidermidis, respectively, were isolated from these PCs. CONCLUSIONS: Between 0.03% and 0.12% of the PCs were contaminated with bacteria. BacT/ALERT is an efficient tool for monitoring PCs for bacterial contamination; however, it is important to realize that false-negative results may occur.     

In-vivo platelet activation correlates with red cell anionic phospholipid exposure in patients with β-thalassaemia major

Several clinical and laboratory findings suggest the presence of a chronic hypercoagulable state in patients with β-thalassaemia major (TM). We have previously shown that isolated TM red blood cells (RBC) strongly enhance prothrombin activation, suggesting an increased membrane exposure of procoagulant phospholipids (i.e. phosphatidylserine). In this study we quantitated the procoagulant activity of RBC in TM and thalassaemia intermedia (TI) patients. We also determined the fraction of activated platelets expressing p-selectin (CD62p) or CD63 in these subjects. Both assays were performed by dual-colour flow cytometry. A significantly ( P  < 0.01) higher fraction of FITC-annexin V-labelled RBC was found in TM and TI patients, compared to the controls. A highly significant correlation ( P  < 0.001) was found in TM patients between the number of RBC-bound annexin V molecules and the fraction of CD62p (p-selectin) or CD63-positive platelets. This association between annexin V binding to TM RBC and the expression of platelet activation markers was also found in individual TM patients over time. Thus, the procoagulant surface of TM RBC may accelarate thrombin generation in vivo which, in turn, triggers platelet activation.     

A novel antigen-specific capture assay for the detection of platelet antibodies and HPA-1a phenotyping

BACKGROUND AND OBJECTIVES: The antigen-specific assays currently used for the laboratory investigation of platelet antibodies and antigens are technically complex and cannot be used in most routine laboratories. Here, we describe a simple antigen-specific capture assay (ASCA) for the detection of serum platelet antibodies and for human platelet antigen-1a (HPA-1a) phenotyping. MATERIALS AND METHODS: For the detection of platelet antibodies, platelets from healthy blood donors were incubated with biotinylated monoclonal antibodies to platelet glycoprotein complexes (GP), then solubilized and mixed with superparamagnetic streptavidin particles. Serum samples from patients with autoimmune thrombocytopenia (n = 39), from patients with platelet alloantibodies (6 HPA-1a, 1 HPA-2b, 1 HPA-3a, 6 HPA-5b), and from healthy blood donors (n = 70), were tested. All serum samples from the patients were investigated in parallel by the indirect monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA). For HPA-1a phenotyping, superparamagnetic particles were coated with a monoclonal antibody to HPA-1a and mixed with diluted whole blood samples from healthy blood donors (n = 139), who had previously been genotyped for platelet alloantigens. Results The indirect MAIPA detected autoantibodies in 18%, and the direct MAIPA in 50% of patients tested. In contrast, the new ASCA demonstrated positive results in 77% of patients. All tested alloantibodies reacted positive by the ASCA, and all serum samples from healthy blood donors were negative. The results of HPA-1a phenotyping were in concordance with those of genotyping in all cases. CONCLUSION: In our opinion, the ASCA is easy to perform and much more sensitive than the currently available antigen-specific assays for the detection of platelet antibodies.