Improved in-vitro quality of platelet concentrates stored in a dextrose-free synthetic medium

Summary. The licenced balanced salt solution Plasma-Lyte, buffered with a clinical solution of sodium bicarbonate, was evaluated as a suspending fluid for platelet concentrates. Platelets suspended in this medium showed better pH maintenance over 5 days of storage compared to platelets stored in plasma (7·0 vs 6·45, P < 0·001). This was reflected in improvements in in-vitro indicators of platelet viability hypotonic shock response (79 vs 48%, P < 0·05), aggregation to paired agonists (86 vs 62%, P < 0·05); and platelet size distribution (104 vs 119%, P < 0·001). Dissolved bicarbonate measurement showed less depletion of bicarbonate in the synthetic medium compared to plasma, which suggests a lower rate of lactate formation. A synthetic medium containing dextrose showed inferior platelet storage characteristics when compared to the plasmalyte/bicarbonate medium in a paired study (Day 5, pH 6·53 vs 6·9, P < 0·05). The results suggest that utilization of substrates other than dextrose allows platelets to metabolize without the accumulation of lactate that leads to pH drops during storage in plasma, and continue to support the feasibility of storing platelets in a non-plasma environment.     

Variation of pH-measurement in platelet concentrates

To measure pH in platelet concentrates, blood gas analysers with different calibration principles may be used. In this study, variances observed in pH measurements with two types of blood gas analysers were investigated. pH was measured in crystalloid solutions (platelet additive solution (PAS-II), phosphate-buffered solutions) and two types of platelet concentrates (containing 100% plasma, or 65% PAS-II/35% plasma) with two blood gas analysers: either using liquid and gas calibration (AVL 945), or only liquid calibration (AVL OMNI). These measurements were compared with a reference method. Especially for PAS-II, large variation in pH was observed between AVL 945, AVL OMNI and the reference method: 6.91 +/- 0.02, 7.35 +/- 0.02 and 7.188 +/- 0.010, respectively (mean +/- SD; n = 12, P < 0.0001, paired t-test). A significant difference in pH was also found for platelet concentrates in 65% PAS/35% plasma (6.88 +/- 0.09 on AVL 945 and 7.02 +/- 0.09 on AVL OMNI, n = 134, P < 0.0001). Comparison with the reference method revealed minor differences with AVL 945, whereas AVL OMNI gave a mean difference in pH of + 0.17. Platelets in 100% plasma revealed smaller differences (6.93 +/- 0.13 for AVL 945 and 6.99 +/- 0.13 for AVL OMNI, n = 95, P < 0.0001). We conclude that different blood gas analysers can yield different pH values, especially in weak buffered solutions such as platelet concentrates in PAS-II. Validation of blood gas analysers for pH measurement of these solutions is therefore mandatory.     

Storage of single donor platelet concentrates: Paired comparison of storage as single or double concentrates

Modern cell separators allow the collection of two plateletpheresis concentrates (PCs) at one session. This study evaluates the quality of PCs stored as double concentrates in standard storage containers of two manufacturers. We collected 20 PCs that contained 4.5 × 10¹¹ platelets in 375 ml plasma (10 using the COBE Spectra and 10 using the Fresenius AS.TEC 204 with 500 ml bags) that were split into one unit of 3.0 × 10¹¹ platelets in 250 ml (3.0‐PC) and one of 1.5 × 10¹¹ platelets in 125 ml (1.5‐PC). Storage of one 3.0‐PC per bag of a two‐bag system corresponded to storage conditions for double PCs and storage of one 1.5‐PC per bag to storage conditions of single PCs. Cell counts, blood gas analysis, glucose and lactate levels, platelet aggregation, and activation and plasma levels of β‐ thromboglobulin (β‐TG) and complement factor 3a (C3a) were measured before storage and again on days 3 and 5. COBE 3.0‐PCs demonstrated less pH rise, lactate production, CD 62P expression and β‐TG plasma levels, and better aggregability after storage than COBE 1.5‐PCs. Fresenius 1.5‐PCs had similar platelet quality to COBE 3.0‐PCs. Fresenius 3.0‐PCs showed a fall of pH (day 5: 6.22 ± 0.56), the highest amount of anaerobic glycolysis compared to all other storage conditions investigated, high CD 62P‐ expression and β‐TG plasma levels, and impaired aggregability on days 3 and 5. The highest C3a levels were found in COBE 1.5‐PCs. 3.0 × 10¹¹ platelets in 250 ml plasma should be stored either in one bag of the COBE system or in two 500 ml bags of the Fresenius system. The COBE two‐bag system allows the storage of two PCs without loss of platelet quality. Two PCs should not be stored in the Fresenius C4L 500 ml storage containers. J. Clin. Apheresis. 16:148‐154, 2000. © 2001 Wiley‐Liss, Inc.     

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).     

Extended storage of granulocyte concentrates mobilized by G-CSF with/without dexamethasone and collected by bag separation method

The aim of this study was to examine the extended storage of granulocyte concentrates mobilized by granulocyte-colony-stimulating factor (G-CSF) with/without dexamethasone (DEX) and collected by a bag separation method. Ten healthy adult volunteers donated blood three times: twice after granulocyte mobilization by (1) injecting G-CSF at 3 microg kg(-1) subcutaneously (s.c.) and (2) injecting G-CSF at 3 microg kg(-1) s.c. + DEX at 8 mg per oral and once (3) for a baseline control without any forms of mobilization. Granulocytes were collected by a bag separation method. The functions (phagocytosis and oxidative killing levels), viability and levels of interleukin (IL)-1beta, IL-8, IL-6 and tumour necrosis factor-alpha of granulocytes were measured. The average numbers of granulocytes collected from 200-mL samples of whole blood from the G-CSF and G-CSF + DEX groups were 35.1 x 10(8) and 49.4 x 10(8), respectively. Phagocytosis level, oxidative killing level and the viability of the granulocytes mobilized by G-CSF with/without DEX were well maintained for up to 72 h of storage after collection. The levels of the cytokines increased in a time-dependent manner. The in vitro phagocytosis level, oxidative killing level and the viability of granulocytes mobilized by G-CSF with/without DEX and collected by bag separation method can be maintained for as long as 72 h after collection.     

Amotosalen photochemical inactivation of severe acute respiratory syndrome coronavirus in human platelet concentrates

A novel human coronavirus causing severe acute respiratory syndrome (SARS) emerged in epidemic form in early 2003 in China and spread worldwide in a few months. Every newly emerging human pathogen is of concern for the safety of the blood supply during and after an epidemic crisis. For this purpose, we have evaluated the inactivation of SARS-coronavirus (CoV) in platelet concentrates using an approved pathogen inactivation device, the INTERCEPT Blood System. Apheresis platelet concentrates (APCs) were inoculated with approximately 10(6) pfu mL(-1) of either Urbani or HSR1 isolates of SARS-CoV. The inoculated units were mixed with 150 microm amotosalen and illuminated with 3 J cm(-2) UV-A light. The viral titres were determined by plaque formation in Vero E6 cells. Mixing SARS-CoV with APC in the absence of any treatment decreased viral infectivity by approximately 0.5-1 log10. Following photochemical treatment, SARS-CoV was consistently inactivated to the limit of detection in seven independent APC units. No infectious virus was detected after treatment when up to one-third of the APC unit was assayed, demonstrating a mean log10-reduction of >6.2. Potent inactivation of SARS-CoV therefore extends the capability of the INTERCEPT Blood System in inactivating a broad spectrum of human pathogens including recently emerging respiratory viruses.     

Improved platelet compatiblity of water vapour glow discharge treated non-woven poly(ethylene terephthalate) leukocyte-reduction filters for different types of platelet concentrates

Non-woven poly[ethylene terephthalate] (NW-PET) filter fabric, usually used for leucocyte removal of red cells, was modified by water vapour glow discharge (WVGD) treatment to improve platelet compatibility. Modified filter material was evaluated with different kinds of platelet concentrates (PCs). In addition, modified filter materials were gamma-sterilized and tested after different time intervals at different storage conditions. Modification of the filter material resulted in an improved platelet recovery after filtration of PC from 57 to about 80%. No significant difference in platelet recovery was observed when filtering either freshly prepared (79 +/- 3.5%, mean +/- SD), overnight-stored single BC-PC (78 +/- 3.3%), overnight-stored single PRP-PC (75 +/- 8.8%) or overnight-stored pooled BC-PC (79 +/- 8.9%). However, freshly prepared pooled BC-PC gave a significantly higher platelet recovery (84 +/- 3.5%). Leukocyte depletion did not differ significantly between the different types of PC. gamma-Sterilization and subsequent storage of the modified filter material for 5, 14 and 26 weeks at 20 degrees C or 37 degrees C had no significant influence on the filtration results of overnight-stored pooled BC-PC. The results of the present study show that WVGD-treated NW-PET is platelet compatible and can be used for leucocyte removal from preferably BC-PC. It can be gamma-sterilized and stored for at least 6 months prior to filtration without affecting the platelet recovery and leucocyte removal.     

Platelet concentrates derived from buffy coat and apheresis: biochemical and functional differences

Today, platelet concentrates are generally produced from whole blood by differential centrifugation (buffy coat-derived platelet concentrates--PCs) or by plateletpheresis (apheresis-derived platelet concentrates--APCs). As PCs are characterized by a lower number of platelets than APCs, four to six PCs are customarily combined in order to obtain an equivalent dose. In the 1970s and 1980s, the use of PCs exceeded that of APCs by far; in contrast, since the beginning of the 1990s, APCs comprise more than half of all transfused platelets. However, the selection of PCs or APCs for transfusion to thrombocytopenic patients is still a matter of debate. The present paper compares biochemical and functional properties of both platelet preparations in vitro. Besides plasma parameters (e.g. platelet factor 4 (PF4), P-selectin, C3a-desarginin, plasma coagulation factors), platelet function was analysed by aggregometry and the PFA 100 system. APCs are characterized by a better preservation of ADP and collagen-induced platelet aggregation, and shorter closure times of the PFA 100 test system during storage. The improved primary in vitro haemostatic capacity of APCs is presumed to be owing to a lower cellular activation rate in these preparations. This hypothesis is supported by the higher plasma concentrations of PF4, beta-thromboglobulin and P-selectin found in PCs compared with APCs. The concentrations of C3a-desarginin in PCs exceed those in APCs by far. Additionally, thrombin generation is higher in PCs than in APCs. These data suggest that APCs are characterized by a superior haemostatic capacity over PCs in vitro. However, in vivo studies should be performed to confirm these findings in the patients' circulation also.     

Platelet storage lesion in interim platelet unit concentrates: A comparison with buffy-coat and apheresis concentrates

Platelet storage lesion is characterized by morphological changes and impaired platelet function. The collection method and storage medium may influence the magnitude of the storage lesion. The aim of this study was to compare the newly introduced interim platelet unit (IPU) platelet concentrates (PCs) (additive solution SSP+, 40% residual plasma content) with the more established buffy-coat PCs (SSP, 20% residual plasma content) and apheresis PCs (autologous plasma) in terms of platelet storage lesions. Thirty PCs (n = 10 for each type) were assessed by measuring metabolic parameters (lactate, glucose, and pH), platelet activation markers, and in vitro platelet aggregability on days 1, 4, and 7 after donation. The expression of platelet activation markers CD62p (P-selectin), CD63 (LAMP-3), and phosphatidylserine was measured using flow cytometry and in vitro aggregability was measured with multiple electrode aggregometry. Higher platelet activation and lower in vitro aggregability was observed in IPU than in buffy-coat PCs on day 1 after donation. In contrast, metabolic parameters, expression of platelet activation markers, and in vitro aggregability were better maintained in IPU than in buffy-coat PCs at the end of the storage period. Compared to apheresis PCs, IPU PCs had higher expression of activation markers and lower in vitro aggregability throughout storage. In conclusion, the results indicate that there are significant differences in platelet storage lesions between IPU, buffy-coat, and apheresis PCs. The quality of IPU PCs appears to be at least comparable to buffy-coat preparations. Further studies are required to distinguish the effect of the preparation methods from storage conditions.     

Transfusion of platelet concentrates from pooled buffy-coats: comparison of bedside vs. prestorage leukofiltration

Prestorage leucocyte filtration of platelet concentrates (PC) has been considered to be superior to bedside filtration with regard to the incidence of nonhaemolytic transfusion reactions (NHTR) or HLA-alloimmunization. It is currently a matter of debate whether prestorage leucocyte filtration has an impact on the storability of platelets and on the transfusion results of PC. In a clinical retrospective study we investigated the transfusion results of PC from pooled buffy-coats (PC-BC) in haematological patients without known refractoriness to platelets, and compared bedside filtration (n = 228/36 patients) vs. prestorage filtration (n = 271/25 patients). Leukocyte and platelet content of the PC, duration of storage, platelet count of the patient before transfusion and 20 h after transfusion were determined and platelet increment and corrected count increment (CCI) 20 h after transfusion were calculated. The mean leucocyte content of the bedside filtered PC was 66 +/- 50 x 106 and < 0.1 x 106 for the prestorage filtered PC (P < 0.001). Mean platelet content of the PC (2.6 x 1011 vs. 2.7 x 1011) and the duration of PC storage (2.7 vs. 2.6 days) were almost identical in both groups. The platelet increment after 20 h (14.6 x 109 L-1 vs. 14.9 x 109 L-1) was equal for both groups, but CCI values were significantly higher for the bedside filtered PC (14.1 +/- 9.5 vs. 11.4 +/- 6.8) (P = 0.008). Correlation with the storage time revealed that CCI levels were higher for bedside filtered PC after short-term storage (< 36 h) (P = 0.014), but declined more rapidly compared with prestorage filtered PC. A patient-based analysis including fewer cases revealed superior but nonsignificant results for bedside filtration. In conclusion, bedside filtered PC-BC resulted in better CCI results after short-term storage, but values equalized with prestorage filtered PC-BC after longer storage intervals. Prestorage-leucocyte filtration did not improve platelet recovery in vivo, but CCI values decreased only moderately throughout storage. Both preparations showed excellent transfusion results even after a 5-day storage interval.     

Platelets do not adsorb HLA class I molecules during storage of pooled platelet concentrates

The origin of HLA class I molecules on platelets is still under discussion. Adsorption of HLA molecules on platelets using specific experimental conditions has been described. The study presented investigates whether there is a significant elution and adsorption of HLA class I molecules on platelets during storage of pooled random platelet concentrates (PRPC) under routine conditions. Platelet concentrates (PCs) from whole blood were prepared from HLA-A2-positive and HLA-A2-negative donors, pooled and stored under routine conditions. In addition, platelets from HLA-A2-negative donors were pelleted and resuspended in cell-free plasma from HLA-A2-positive donors. HLA-A2-positive PCs (positive control), HLA-A2-negative PCs (negative control) and HLA-A2-negative platelets in plasma from HLA-A2-negative donors were stored simultaneously. Binding of FITC-conjugated monoclonal murine antihuman HLA-A2 antibodies (anti-HLA-A2-mab) was measured during 5-day storage by flow cytometry. An increased binding of anti-HLA-A2-mab during storage was found on HLA-A2-negative platelets (P < 0.005) independently whether they were incubated with cell-free plasma or platelets from HLA-A2-positive donors or autologous HLA-A2-negative cell-free plasma. However, non-specific binding of IgG controls increased equally, whereas anti-HLA-A2-mab binding to platelets from HLA-A2-positive donors did not decrease during storage. This study suggests that there is no significant elution and adsorption of HLA class I antigens of platelets in pooled PCs during storage under the usual conditions for platelet storage. Increased anti-HLA-A2-mab signal was due to non-specific binding. Therefore, HLA class I compatible platelets should maintain their compatibility for an immunized patient when stored in a pool with HLA incompatible platelets and shortened survival after transfusion should not be expected.     

Comparison of two platelet additive solutions

The use of an additive solution for substitution of plasma for storage of leukodepleted platelet concentrates can have many advantages. In this study, a comparison was made between two platelet additive solutions: one containing citrate and acetate (PAS-II), the other also supplemented with additional salts such as magnesium, and with gluconate (Composol-PS). Donor-dependent differences were avoided by applying a paired experimental design (n = 10). The platelet concentrates were prepared by pooling five buffy coats and the additive solution, and prestorage filtration was utilized to remove leucocytes to well below 1 x 106. Storage of platelet concentrates up to 9 days after blood collection revealed that platelet concentrates in Composol-PS maintained an almost constant pH of on average 6.93 from day 2 through day 7, and at 6.90 at day 9. This was in contrast to PAS-II, which showed a gradually decreasing pH from on average 6.97 at day 1 to 6.86 at day 9. In all units stored in both solutions the swirling effect was present during 9 days of storage. In conclusion, both additive solutions allow storage of platelets, derived from pooled buffy coats, for up to 9 days after collection of the whole blood, with maintenance of good quality in vitro. Composol-PS has a slightly better buffering capacity, reflected as a more constant pH throughout the storage period.     

Donor platelet and leukocyte count as predictive factors of the quality of platelet concentrates obtained from whole blood by semiautomated fractionation

Platelet concentrates (PCs) obtained from whole blood are produced by fractionation of the buffy coat (BC) or the platelet-rich plasma. Despite the improvements in the technologies used for the hemocomponent fractionation, the proportion of PCs that do not accomplish the quality requirements is high. This study aimed to determine whether the basal platelet and leukocyte counts are predictive factors of the quality of the PCs obtained from BC by semiautomated fractionation. Quality control registers of 196 PCs were analyzed. Gender- and age-dependence of the blood cell count and the characteristics of PCs were evaluated. Platelet yield and residual leukocytes in the PCs were correlated with the platelet and leukocyte counts and the age of the donors. Predictive efficacy was assessed, and an optimal cut-off was established. The proportions of PCs accepted and rejected by using or not the optimal cut-off were compared. 50.0% of the PCs accomplished all the quality control requirements. Female donors had a higher basal platelet count than males. A correlation was observed between basal platelets and platelet yield, but not between basal leukocytes and residual leukocytes. The basal platelet count predicted the quality of the PCs. A cut-off of 231,000 platelets/mm³ was established, but it did not improve the proportion of accepted PCs. In conclusion, we found that the basal platelet count is correlated with the platelet yield. The basal leukocyte count is not correlated with the residual leukocytes. The established cut-off for the basal platelet count did not improve the proportion of accepted PCs.     

医用电解质溶液作添加液制备汇集少白细胞血小板

目的采用医用电解质溶液作添加液(PAS),建立1种白膜法制备添加液汇集少白细胞血小板(PAS汇集BC-PCs)的方法。方法从400ml全血中分离白膜层,容量35—40ml,放(22±2)℃静置过夜,将ABO同型的6袋白膜汇集,加220g添加液(90%复方电解质注射液、8%ACD-A血液保养液和2%含量为50g/L的碳酸氢钠注射液的混合液)稀释白膜,汇集后的白膜在(22±2)℃中,以300×g离心10min,将上层的富含血小板悬液再经白细胞滤除器过滤去除白细胞,并转移到血小板保存袋内,即制备成1个成人治疗量的PAS汇集BC-PCs,制备过程在一个特制的密闭系统内完成。结果共制备30个成人治疗量的PAS汇集BC-PCs,其容量为(270±32)ml、血小板含量为(2.96±0.31)×1011、WBC混入量为(1.3±0.2)×106、RBC混入量为(5.8±1.1)×109、CD62P表达率为(22.5±10.6)%。保存8d后的pH为7.14±0.04、低渗休克反应率(HSR)为(54.0±8.2)%、CD62P表达率为(45.7±13.8)%。结论由复方电解质注射液、ACD-A保养液和碳酸氢钠注射液的混合液为添加液汇集血小板的方法可行。     

Evaluation of two detection methods of microorganisms in platelet concentrates

Background: The performance of a bacterial 16S ribosomal DNA real‐time polymerase chain reaction (PCR) assay was evaluated and validated with an automated culture system to determine its use for screening of platelet concentrates (PCs). Study Design and Methods: PCs were spiked with suspensions of Escherichia coli, Serratia marcescens, Staphylococcus epidermidis and St. aureus at 1, 10, and 100 colony‐forming units (CFUs) mL and stored for 5 days. DNA amplification was performed using real‐time PCR. The BacT/ALERT was used as a reference method and samples were inoculated into an aerobic culture bottle; for the PCR assay, aliquots were drawn from all (spiked) PCs on days 0 to 5 of storage. Results: Real‐time PCR detected only the gram‐positive bacteria in PCs spiked with low bacterial titres (1 CFU mL) after 48 h; however, it was able to detect all positive samples in PCs spiked with 10 CFU mL of either gram‐positive or gram‐negative bacteria after 48 h. In addition, real‐time PCR detected all positive samples in PCs spiked with high gram‐positive bacterial titres (100 CFU mL) after 24 h. On the other hand, the BacT/ALERT system showed positive results in all samples within 24 h. Conclusion: The BacT/ALERT method is more sensitive and should continue to be the gold standard for identifying bacterial contaminations in blood samples. The real‐time PCR approach can be used for the screening of PCs for microbial detection before they are released from blood centres or shortly before they are used in blood transfusion, and thus allow an extended shelf life of the platelets.