Preparation Of Platelet Concentrates Research Articles

004 Novel Platelet Concentrate Preparation System for Growth Factor Mediated Wound Healing

A promising approach for advanced wound healing involves the use of growth factors from autologous platelet concentrate (PC). A novel PC (NPC) preparation technique, based on platelet aggregation and filtration, has been developed to concentrate platelets (in less than 5 minutes) in a fully disposable system, eliminating the need for a centrifuge or cell saver.NPC was compared with centrifugation‐based PC (CPC) with regard to: 1) platelet recovery, 2) growth factor content, 3) cell proliferative activity with monkey epithelial cells and bone‐marrow mesenchymal cells (BMMC), 4) healing response of Porcine full‐thickness wounds.There was no statistically significant difference in platelet count, growth factor content (platelet‐derived growth factor enzyme‐linked immunosorbent assay), and cell proliferation activity (Fig. 1) between NPC and CPC. Quantitative assays (Alkaline phosphatase and 5‐bromo‐2‐deoxyuridine) in BMMCs also showed that NPC was equal to CPC in its mitogenic activity (n = 6, paired t‐test). Images showing similar cell growth with NPC and CPC.imageThe histopathology of porcine full‐thickness wounds (at 24 days) showed that NPC and CPC (in a hydrogel carrier) treated wounds healed similarly. The NPC and CPC treatments resulted in less scarring, less inflammation, and improved vascularity than hydrogel alone.The NPC’s growth promoting ability is equivalent to CPC, and the technique is significantly simpler, faster, and more economical than CPC. Thus, NPC could dramatically expand the use of PC for wound healing when combined with various wound dressings and adjuvants.

Novel treatment modalities: new platelet preparations and substitutes.

Keywords: platelets; platelet substitutes; platelet haemostatic function; blood substitutes; platelet function

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

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. 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. 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). 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.

Preparation of Leukocyte–Poor Platelet Concentrates via a Short, Hard Spin of a Pool of Buffy Coats

Background and Objectives: A new method for the preparation of leukocyte–poor platelet concentrates was developed, based on a short, hard spin of a pool of 5 buffy coats (BCs) combined with automated collection of the platelets. Materials and Methods: The characteristics of platelet concentrates (PCs) were studied as a function of the total g force applied to a pool of 5 BCs. Pools of BCs were centrifuged for 1 min with a total g force ranging from about 3,300 to 5,000 gmin (n = 7–9 per applied g force). Deceleration took place without the means of a brake. The total centrifugation time was about 11 min. The platelet–rich plasma (PRP) fraction above the cell layer was separated by an automated component preparation device. Results: A short, hard spin with a total g force of between 3,400 and 4,600 gmin resulted in PCs that contained on average more than 290×10⁹ platelets and less than 5×10⁶ leukocytes without the use of a leukocyte filter, provided that the transfer of PRP was electronically checked and terminated. The cell concentrations in the PCs are a function of the total g force. Both the platelet and leukocyte levels in the concentrate decreased with an increase in the total g force applied to the pool. Conclusion: The preparation of PCs via a short hard, spin of BCs, combined with automated collection of the PRP, may be an alternative method for the preparation of leukocyte–poor PCs.

Use of prostaglandin E1 during preparation of platelet concentrates.

A paired study in 10 autologous volunteer donors was undertaken to investigate the efficacy of adding prostaglandin E1 (PGE1) in vitro during routine platelet concentrate (PC) production. After 5 days storage, PCs prepared with PGE1 were compared with control PCs. In vivo platelet recovery, survival and biodistribution were determined following autologous infusion of indium-111 labelled platelets into volunteers, together with the in vitro evaluation of platelet function and biochemistry. PGE1 facilitated easier and faster platelet resuspension following centrifugation. After storage there were few significant in vitro differences between PCs prepared with PGE1 and control PCs. The artifactual leucocyte concentration was significantly lower in the presence of PGE1, suggesting less platelet aggregates had been formed during storage and beta-thromboglobulin release was significantly reduced by PGE1, 14.0 +/- 6.0 micrograms per 10(9) platelets compared with 22.3 +/- 9.8 micrograms per 10(9) platelets in control PCs, (P < 0.01), indicating PGE1 reduced both platelet aggregation and activation probably at the initial preparation stage, known to produce the greatest trauma. Initial in vivo platelet recovery for PCs prepared with PGE1 was similar to that of control PCs, 41.1 +/- 12.5% vs. 44.4 +/- 8.0%, respectively, and there were no differences in organ distribution at 24 h. However, in vivo multiple hit survival was reduced in the presence of PGE1, 5.8 +/- 1.6 days compared with 6.9 +/- 1.4 days in control PCs (P < 0.05). Despite the ability of PGE1 to facilitate platelet resuspension and inhibit platelet aggregation and activation during preparation of the PCs, the reduced in vivo survival time may preclude the use of PGE1 during routine PC preparation.

Growth factor release during preparation and storage of platelet concentrates.

The platelet content of platelet-derived growth factor (PDGF), a mitogen stored in the alpha-granules, was studied during preparation and storage of platelet concentrates (PC) and compared to the growth-promoting activity of platelets, beta-thromboglobulin (beta-TG) and lactate dehydrogenase (LD). We compared PC prepared from platelet-rich plasma (PRP-PC; n = 10) and from buffy coat. Two different pre-preparation storage periods of the buffy coat were used: 4 h (BC-PC:4h; n = 10) and 24 h (BC-PC:24h; n = 5). The platelet content of PDGF and beta-TG was measured by a RIA technique and the growth-promoting activity by incorporation of 3H-thymidine in stimulated fibroblasts. The platelet content of PDGF, beta-TG and the growth-promoting activity of the platelets decreased in a similar way during preparation and storage of PRP-PC (31 +/- 2, 35 +/- 2 and 33 +/- 7%, respectively, at day 5 of storage; mean +/- SEM). The release of LD was minor (3.9 +/- 0.5% at day 5). At day 1 of storage the platelet content of PDGF was significantly better preserved in BC-PC:4h than in BD-PC:24h (88 +/- 2 and 81 +/- 3%, respectively; p = 0.03). Comparing BC-PC:4h and PRP-PC we found a significantly better preservation of PDGF in BC-PC:4h until day 3 of storage (80 +/- 2 and 75 +/- 1%, respectively at day 3; p = 0.046). In conclusion the preparation of PC according to the PRP method initially induces a higher loss of PDGF, and hence of the growth-promoting activity, than the BC method.

Growth Factor Release during Preparation and Storage of Platelet Concentrates

The platelet content of platelet-derived growth factor (PDGF), a mitogen stored in the alpha-granules, was studied during preparation and storage of platelet concentrates (PC) and compared to the growth-promoting activity of platelets, β-thromboglobulin (β-TG) and lactate dehydrogenase (LD). We compared PC prepared from platelet-rich plasma (PRP-PC; n = 10) and from buffy coat. Two different pre-preparation storage periods of the buffy coat were used: 4 h (BC-PC:4h; n = 10) and 24 h (BC-PC: 24h; n = 5). The platelet content of PDGF and β-TG was measured by a RIA technique and the growth-promoting activity by incorporation of 3H-thymidine in stimulated fibroblasts. The platelet content of PDGF, β-TG and the growth-promoting activity of the platelets decreased in a similarway during preparation and storage of PRP-PC (31±2, 35±2 and 33±7%, respectively, at day 5 of storage; mean ± SEM). The release of LD was minor (3.9±0.5% at day 5). At day 1 of storage the platelet content of PDGF was significantly better preserved in BC-PC:4h than in BD-PC:24h (88±2 and 81±3%, respectively; p = 0.03). Comparing BC-PC:4h and PRP-PC we found a significantly better preservation of PDGF in BC-PC:4h until day 3 of storage (80±2 and 75±1%, respectively at day 3; p = 0.046). In conclusion the preparation of PC according to the PRP method initially induces a higher loss of PDGF, and hence of the growth-promoting activity, than the BC method.

Influence of a 12‐hour, 22°C holding period for buffy coats on the preparation of platelet concentrates stored in plasma

The preparation of platelet concentrates (PCs) from buffy coats (BCs) stored at room temperature is controversial, because of the strong metabolic activity of cells in BCs and the possible detrimental effect of neutrophil enzymes on platelets when the holding time before separation is prolonged. Despite good in vitro and in vivo behavior of BC-PCs stored in synthetic solution, little is known of the quality of BC-PCs stored in plasma. Comparison was made of PCs prepared from BCs held at 22 degrees C for 3 hours (3-hour BC-PCs) or overnight (12-hour BC-PCs) and stored in plasma. Platelet and white cell counts, pH, response to osmotic shock, and morphologic scores were determined on 20 PCs of each type. The decrease in dense granule and alpha granule content, a marker of platelet activation, were estimated by mepacrine counting and beta-thromboglobulin measurement, respectively (n = 8-10). Platelet function was studied in terms of aggregation and thromboxane production in response to various concentrations of collagen and thrombin (n = 8-17). PCs prepared from unstored BCs (n = 15) and from BCs held for 90 minutes (n = 15) were used as controls. Platelet yield was increased from 53 +/- 10 percent of donated platelets to 73 +/- 4 percent by increasing the BC holding time from 0 to 90 minutes to 3 hours (p < 0.001). Similar yields (7.8 +/- 1.8 vs. 7.9 +/- 2 x 10(10) platelets) and white cell contamination (0.9 +/- 0.8 vs. 1.0 +/- 0.9 x 10(7)) were obtained with 3-hour and 12-hour BC-PCs. At the end of the storage period (Day 5), all variables known to correlate with platelet survival in vivo were well maintained in both 3-hour and 12-hour BC-PCs: pH > or = 6.9, response to osmotic shock > or = 70 percent, and morphology scores always > or = 240. During storage, the dense granule content decreased moderately (30% after 5 days), whatever the conditions. By contrast, the total platelet beta-thromboglobulin content was better preserved in 12-hour BC-PCs than in 3-hour BC-PCs (p < 0.04). No significant differences were observed in collagen-induced aggregation and thromboxane production in the two PC preparations. However, aggregation responses to thrombin were higher in 12-hour BC-PCs on Day 5 of storage (p < 0.01). BCs can be held at 22 degrees C for up to 12 hours, with no detrimental effect on the quality of PCs stored for up to 5 days in plasma. Such a holding time might help overcome logistic problems in blood banks.

In vitro evaluation of a new filter for leucocyte depletion of platelet concentrate during component preparation.

A newly developed filter for prestorage leucocyte depletion of platelet concentrates (PC) was studied. The filter is designed for leucocyte depletion during the preparation of the pool PC from platelet rich buffy coats. In all the leucocyte depleted PC (LD-PC) leucocyte depletion was satisfactory. 19 of 20 units of LD-PC had a leucocyte content below 3 x 10(5) per PC and 1 contained 8 x 10(5) leucocytes. The standard PC contained 2.53 x 10(8) (0.87 x 10(8)-15.3 x 10(8); n = 20) leucocytes per PC (median and range). The quality of the LD-PC was evaluated by measuring platelet activation, platelet morphology, and pre- and poststorage pH. There were no differences in any of the parameters studied.

Effect of Prestorage Leukocyte Removal on the Cytokine Levels in Stored Platelet Concentrates

The effect of the prestorage removal of leukocytes from platelet concentrates (PC) on the cytokine levels during its storage was studied. Two methods for leukocyte removal were examined: filtration and preparation of the PC by the buffy coat method. Cytokine levels were measured at various storage times. Highly increased levels of tumor necrosis factor-alpha (TNF-alpha; 120 +/- 131 ng/l) and interleukin 6 (IL-6; 988 +/- 494 ng/l) were found after a 5-day storage in the control group, whereas no increased levels were found in filtered PC (TNF-alpha 14 +/- 4 ng/l, IL-6 < 4 ng/l) or in buffy coat PC (TNF-alpha 8 +/- 2 ng/l, IL-6 < 4 ng/l). Furthermore an effect of the pooling of buffy coats or PC on the cytokine levels was not found. Transfusion of PC containing high levels of IL-6 and TNF-alpha has been associated with febrile transfusion reactions in the recipient and therefore the prestorage leukocyte removal might prevent these febrile transfusion reactions. The preparation of buffy coat PC, through its simplicity, seems to be the method of choice.

In vitro Evaluation of a New Filter for Leucocyte Depletion of Platelet Concentrate during Component Preparation

A newly developed filter for prestorage leucocyte depletion of platelet concentrates (PC) was studied. The filter is designed for leucocyte depletion during the preparation of the pool PC from platelet rich huffy coats. In all the leucocyte depleted PC (LD-PC) leucocyte depletion was satisfactory. 19 of 20 units of LD-PC had a leucocyte content below 3x10^5 per PC and 1 contained 8x10^5 leucocytes. The standard PC contained 2.53x10^8 (0.87x10^8-15.3x10^8; n = 20) leucocytes per PC (median and range). The quality of the LD-PC was evaluated by measuring platelet activation, platelet morphology, and pre- and poststorage pH. There were no differences in any of the parameters studied.

Role of lactate in platelet storage lesion.

It is known that lactate accumulation may cause a pH fall in platelet concentrates (PC) during storage, and this phenomenon causes platelet morphological lesions and loss of platelet in vivo viability. In this study, we added increasing amounts of lactate to identical PC in order to evaluate the role of hydrogen ion accumulation in determining platelet activation and lesion during storage. Six hours after PC preparation, lactate was added to PC1 and PC2 at 20 and 12 mM final concentrations, respectively, while PC3 served as control. In PC1, pH was lower than 6.3, and platelet function and discoid morphology were lost. PC2 were stored for 7 days at pH values ranging from 6.4 to 6.6, and most results of in vitro measurements reflecting platelet function such as osmotic reversal, ATP release and aggregation in response to different stimuli were not significantly inferior when compared to controls. The addition of lactate had no apparent effect on the rise of platelet activation markers P-Selectin, lysosome-like protein gp 53, platelet-bound fibrinogen and granulophysin, while a reduction of borderline significance was observed in glycoprotein Ib expression after pH reduction to values lower than 6.6. It is concluded that the rise of platelet activation markers during storage reflects platelet lesions different from those determined by lactate per se.

Role of Lactate in Platelet Storage Lesion

It is known that lactate accumulation may cause a pH fall in platelet concentrates (PC) during storage, and this phenomenon causes platelet morphological lesions and loss of platelet in vivo viability. In this study, we added increasing amounts of lactate to identical PC in order to evaluate the role of hydrogen ion accumulation in determining platelet activation and lesion during storage. Six hours after PC preparation, lactate was added to PCI and PC2 at 20 and 12 mM final concentrations, respectively, while PC3 served as control. In PCI, pH was lower than 6.3, and platelet function and discoid morphology were lost. PC2 were stored for 7 days at pH values ranging from 6.4 to 6.6, and most results of in vitro measurements reflecting platelet function such as osmotic reversal, ATP release and aggregation in response to different stimuli were not significantly inferior when compared to controls. The addition of lactate had no apparent effect on the rise of platelet activation markers P- Selectin, lysosome-like protein gp53, platelet-bound fibrinogen and granu- lophysin, while a reduction of borderline significance was observed in glycoprotein lb expression after pH reduction to values lower than 6.6. It is concluded that the rise of platelet activation markers during storage reflects platelet lesions different from those determined by lactate per se.

Amphotericin B‐induced injury in stored human platelets

Administration of intravenous amphotericin B is a major factor in the reduction of the recovery of transfused platelet concentrates (PCs). For the study of possible drug-induced platelet lesions, fresh platelets and stored PCs were incubated with therapeutic concentrations of amphotericin B (10-50 micrograms/mL) and examined for membrane changes by scanning electron microscopy. Stored platelets demonstrated the formation of "pits" on the surface membrane, which were maximal immediately after preparation and gradually decreased with storage. The number of pits was significantly higher after exposure to amphotericin B. No increase was detected in fresh platelets following exposure to amphotericin B. The maximal effect of the drug was seen after 5 days of storage as PCs. Amphotericin B did not affect platelet shape or the number of pseudopodia. There was no correlation between pit formation and the pH, pO2, or pCO2 of the concentrates. Amphotericin B did not release 51Cr from prelabeled stored platelets after 2 hours' incubation at 37 degrees C. Thus, amphotericin B appears to exacerbate a membrane lesion induced by the preparation and storage of PCs.

Vesiculation of platelets during in vitro aging

Membranous microparticles (MP) appearing in the supernatant plasma of stored platelet concentrates (PC) were analyzed by flow cytometry. Two populations of MP were arbitrarily delineated by light scatter as larger or smaller than 0.5 micron fluorescent beads. An estimate of MP concentration was obtained by adding a known amount of fluorescent beads to each sample before analysis of a set number of counts on the flow cytometer. The addition of platelet activation inhibitors (prostaglandin E-1, theophylline, and aprotinin) to the anticoagulant during preparation of PC combined with a reduction in surface area of the storage container caused approximately a 40% reduction in the number of MP appearing during storage relative to donor-matched controls. In addition, the inhibited concentrates had 84% less platelet factor 3 (PF3) activity in the supernatant and 61% less released lactic dehydrogenase. A reduction in surface area of the container in the controls partially offset these differences. A significant correlation was found (rs = .748) between PF3 levels and the concentration of larger MP. The inhibitors did not reduce the small number of MP found in stored platelet-poor plasma. Surface antigen analysis showed that the majority of MP in PC were platelet-derived; most were positive for glycoprotein (GP) IIbIIIa (73%) and/or for GPIb (43% to 46%). We conclude that procoagulant MP are released from platelets during storage as a result of platelet activation augmented by interaction of platelets with the bag wall.

Vesiculation of platelets during in vitro aging

Membranous microparticles (MP) appearing in the supernatant plasma of stored platelet concentrates (PC) were analyzed by flow cytometry. Two populations of MP were arbitrarily delineated by light scatter as larger or smaller than 0.5 micron fluorescent beads. An estimate of MP concentration was obtained by adding a known amount of fluorescent beads to each sample before analysis of a set number of counts on the flow cytometer. The addition of platelet activation inhibitors (prostaglandin E-1, theophylline, and aprotinin) to the anticoagulant during preparation of PC combined with a reduction in surface area of the storage container caused approximately a 40% reduction in the number of MP appearing during storage relative to donor-matched controls. In addition, the inhibited concentrates had 84% less platelet factor 3 (PF3) activity in the supernatant and 61% less released lactic dehydrogenase. A reduction in surface area of the container in the controls partially offset these differences. A significant correlation was found (rs = .748) between PF3 levels and the concentration of larger MP. The inhibitors did not reduce the small number of MP found in stored platelet-poor plasma. Surface antigen analysis showed that the majority of MP in PC were platelet-derived; most were positive for glycoprotein (GP) IIbIIIa (73%) and/or for GPIb (43% to 46%). We conclude that procoagulant MP are released from platelets during storage as a result of platelet activation augmented by interaction of platelets with the bag wall.

The preparation of platelet concentrates by the light-spin/ hard-spin technique

For at least two decades the light-spin/hard-spin (LS/HS) method for preparation of platelet concentrates (PC) has been the standard of platelet support. With concern over the detrimental effects of platelet activation during component preparation and with increased recognition of the adverse consequences resulting from residual donor leukocytes in PC, new approaches to the production of PC have begun. This review addresses two aspects of the traditional LS/HS method of platelet preparation: platelet activation and residual leukocyte content. Studies of platelet activation are reviewed which focus on the second (hard-spin) centrifugation step during which pelleting of platelets occurs. Platelets studied immediately after the hardspin exhibit evidence of α-granule release, expression of activation antigens, and decreased aggregation. There is a suggestion that some degree of reversal of platelet activation routinely occurs during the rest period following the hard-spin. The residual leukocyte content of PC prepared by the LS/HS method ranges from 10 7 to 10 9 leukocytes/unit. The residual donor leukocytes are predominantly lymphocytes and monocytes. Degeneration of residual donor leukocytes may release soluble cytokines resulting in febrile transfusion reactions. It remains controversial whether or not the cell-membrane fragments and microvesicles of degenerating donor leukocytes are capable of HLA allosensitization or viral transmission. Release of leukocyte elastase from degenerating leukocytes during platelet storage has been proposed as contributing to the platelet storge lesion. More research is needed to address the question of whether or not pre-storage leukocyte reduction during component preparation will result in improved PC. It appears likely that within the next few years radical changes will occur in the method of preparation of PC with the aim of providing the greatest degree of hemostatic effectiveness with the least toxicity to patients.

Concepts About Current Conditions for the Preparation and Storage of Platelets

Substantial experimentation over the last 20 years has led to the conditions that are currently used to prepare and store platelets. Although platelet rich plasma is used in most instances to prepare platelet concentrates, there may be some benefit associated with the use of buffy coats as the source component. Extension of the maximum allowable storage time for platelets to 5 days has been possible as a result of defining the conditions which allow for the better retention of platelet properties. Storage temperature, permeability of the storage container, volume of platelet suspension, and the need to agitate platelets have been identified as key parameters that maintain platelet viability and functional properties. Storage in the 20 to 24 degrees C range prevents a reduction in posttransfusion viability that occurs when platelets are maintained at lower temperatures. Adequate influx of oxygen through container walls to support platelet metabolism and, to a lesser degree, adequate efflux of produced carbon dioxide are essential for maintaining pH levels, a key parameter that also influences posttransfusion viability. Permeability is influenced by container size and material, by use of a satisfactory volume of plasma and by agitating the container. Although platelet concentrates prepared from whole blood have been primarily used to delineate appropriate storage conditions, they also apply to platelets harvested by aphersis technology. Storage under currently used conditions, although providing products with acceptable clinical efficacy, is associated with a reduction in viability and functional characteristics. The development of storage media, specific for platelets, may minimize the occurrence of deleterious changes.

Platelet Concentrates in an Additive Solution Prepared from Pooled Buffy Coats.

Abstract. A new method for the preparation of platelet concentrates (PCs) is described. The source material is buffy coat (BC), prepared after keeping standard CPD whole‐blood units at room temperature for 6–12 h, followed by centrifugation at 3,500 rpm for 10 min (first series) or 4,000 rpm for 12.5 min (second series). BC, separated from plasma and red cells, was kept at room temperature for a further 8–12 h without agitation. Pools of 6 (first series) and 4 (second series) BCs were prepared using a sterile docking device and suspended in a platelet‐additive solution (PAS) containing sodium/potassium chloride, citrate, phosphate, and mannitol. After gentle centrifugation, the platelet‐rich supernatant was expressed to and stored in one (first series) or two (second series) 1‐liter polyolefine (PL‐732) containers. In the first series, the total number of platelets was 316 ± 59 times 109 per PC (yield 65%). However, when the method was applied at a routine scale, the yield varied considerably and was shown to be strongly dependent on the hematocrit of the BCs. A number of steps were taken to standardize the technique which resulted in an improved yield (77.3 ± 8.7%) with 316 ± 52 times 109 platelets (mean ±SD, range 203–490, n = 134), obtained from 4 BC pools and lower leukocyte contamination than before, 18 ±17 times 106 per preparation (range 1–73, microscopic counting, n = 38). The storage medium consisted of a mixture of plasma and PAS. Standardization of the hematocrit and volume of the BCs is essential, both for improvement of the yield and for providing a sufficient amount of glucose and bicarbonate to the platelets throughout storage. The method is particularly suitable in combination with automated techniques for blood component preparation which include BC removal. Blood gases, pH and adenosine triphosphate were maintained at satisfactory levels; the release of platelet factor 4 and lactate dehydrogenase was not different from traditional PC preparation and storage. However, the content of glucose was exhausted in many PC units towards the end of a storage of 6 days. When whole blood and BC are stored for such a long period, and when the gas permeability of their containers is poor, it is important, in order to avoid early loss of swirling, that the surface‐to‐volume ratio and the gas permeability of the PC containers is sufficiently large to allow good gas exchange.

Platelet Concentrates in an Additive Solution Prepared from Pooled Buffy Coats

A new method for the preparation of platelet concentrates (PCs) is described. The source material is buffy coat (BC), prepared after keeping standard CPD whole-blood units at room temperature for 6-12 h, followed by centrifugation at 3,500 rpm for 10 min (first series) or 4,000 rpm for 12.5 min (second series). BC, separated from plasma and red cells, was kept at room temperature for a further 8-12 h without agitation. Pools of 6 (first series) and 4 (second series) BCs were prepared using a sterile docking device and suspended in a platelet-additive solution (PAS) containing sodium/potassium chloride, citrate, phosphate, and mannitol. After gentle centrifugation, the platelet-rich supernatant was expressed to and stored in one (first series) or two (second series) 1-liter polyolefine (PL-732) containers. In the first series, the total number of platelets was 316 ± 59 x 10(9) per PC (yield 65%). However, when the method was applied at a routine scale, the yield varied considerably and was shown to be strongly dependent on the hematocrit of the BCs. A number of steps were taken to standardize the technique which resulted in an improved yield (77.3 ±8.7%) with 316± 52x10(9) platelets (mean ± SD, range 203-490, n = 134), obtained from 4 BC pools and lower leukocyte contamination than before, 18 ± 17xl0(6) per preparation (range 1-73, microscopic counting, n = 38). The storage medium consisted of a mixture of plasma and PAS. Standardization of the hematocrit and volume of the BCs is essential, both for improvement of the yield and for providing a sufficient amount of glucose and bicarbonate to the platelets throughout storage. The method is particularly suitable in combination with automated techniques for blood component preparation which include BC removal. Blood gases, pH and adenosine triphosphate were maintained at satisfactory levels; the release of platelet factor 4 and lactate dehydrogenase was not different from traditional PC preparation and storage. However, the content of glucose was exhausted in many PC units towards the end of a storage of 6 days. When whole blood and BC are stored for such a long period, and when the gas permeability of their containers is poor, it is important, in order to avoid early loss of swirling, that the surface-to-volume ratio and the gas permeability of the PC containers is sufficiently large to allow good gas exchange.