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Preparation of Leucocyte-Poor Platelet Concentrates from Buffy Coats
Abstract
To study survival and function of leukocyte-poor platelet concentrates (lp-PC) prepared from buffy coats, random platelet transfusions requested for thrombocytopenic patients were evaluated. The lp-PC issued were stored at 22 degrees C for either 1, 3 or 5 days before transfusion. From 31 transfusions, posttransfusion corrected count increments (CCI), corrected for body surface (m2) and divided by number of platelets transfused (1011), were calculated. The mean +/- SEM of the 1-, 24- and 48- hour CCI was 12.2 +/- 0.45, 11.2 +/- 0.51 and 8.8 +/- 0.58, respectively. No significant differences in CCI 24 h after transfusion were observed for 1p-PC stored for 1, 3 or 5 days. Hemostatic activity was observed in all 9 evaluable patients. It is concluded that platelets from 1p-PC survive well in patients, regardless of storage for 1, 3 or 5 days and that the platelets are hemostatically active after transfusion.
... The same Dutch group reported that it is possible to delay whole blood separation up to 24 h if units are cooled to 22°C within about 2 h after collection, without an excessive loss of plasma factor VIII activity (decrease of over 20%), RBC in vitro properties or significant loss of platelet recovery. The only parameter negatively affected by the overnight holding of whole blood at 22°C was RBC 2,3-diphosphoglycerate (DPG) concentration [17]. However, the holding of whole blood bag overnight at 22°C, led to concerns about bacterial overgrowth. ...
Platelet rich plasma (PRP) method was the first method used for platelet concentrate (PC) preparation and continues to be the only licensed method in the USA. By contrast in Europe since the eighties of the last century, the PRP method has been progressively replaced by methods based on the removal of the buffy coat (BC) layer. Initially BC were processed individually to produced a single PC but it was soon recognized that the pooling of 4–6 BC units with the addition of a plasma unit of one of the donors of the pool or a platelet additive solution (PAS), improved the efficiency of the separation and eased the transfusion process since the final product was stored as an adult transfusion dose ready to be connected to the patient. Semi-automatic devices were developed for the separation of whole blood into red blood cells, plasma and the BC and for the separation of the BC into the PC. The introduction of PAS in combination with plasma (in general about 35% of plasma and 65% PAS) for storing PC has shown to offer advantages such as an increased availability of plasma for fractionation, a reduction of transfusion reactions to platelets and a potential reduction in the risk of transfusion related acute lung injury. While early generations of PAS were associated to a decrease in the post-transfusion corrected platelet count in comparison to PC stored in 100% plasma, this effect has not been found with the newer generation of PAS.
... (1) In the BC method, the whole blood is hard spun, the supernatant PPP is removed, the BC is transferred to another bag, the BC is soft spun, and the supernatant platelets are removed to a storage bag. (2) In most blood centers, BCs from 4 to 6 whole blood collections are pooled before the soft spin of the BC is performed, and the BCs are stored as pre-storage pools. (3) Most blood centers in Europe prepare PC as BC, and Canada has recently adopted this system. ...
Platelet (PLT) concentrates (PCs) prepared from whole blood in the United States are made using the PLT-rich plasma method. The PCs must be made within 8 hours of blood collection and stored for only 5 days. In Europe and Canada, PCs are made using the buffy coat (BC) method from whole blood held overnight at 22°C and storage times may be up to 7 days. Our studies were designed to determine how long BC PLTs can be stored in plasma or Plasmalyte while meeting the FDA's poststorage viability criteria.
Normal subjects donated whole blood that was stored at 22°C for 22 ± 2 hours before preparation of BC PLTs. PLTs were stored for 5 to 8 days in either plasma or Plasmalyte concentrations of 65 or 80%. Radiolabeled autologous stored versus fresh PLT recoveries and survivals were assessed as well as poststorage in vitro assays.
BC PLTs stored in either plasma or 65% Plasmalyte met FDA poststorage PLT recovery criteria for 7 days but survivals for only 6 days, while storage in 80% Plasmalyte gave very poor results. Both stored PLT recoveries and survivals correlated with the same donor's fresh results, but the correlation was much stronger between recoveries than survivals. In vitro measures of extent of shape change, morphology score, and pH best predicted poststorage PLT recoveries, while annexin V binding best predicted PLT survivals.
BC PLTs stored in either plasma or 65% Plasmalyte meet FDA's poststorage viability criteria for 6 days.
The buffy coat method as a source for platelet concentrates was developed in the 1970s and is still used in many blood centres around the world. Development of the method sparked various technological advances in blood collection, processing and storage. At the time, the need for platelet concentrates sharply increased because of better treatment regimens for (onco)haematological dises, which forced blood centres to standardize and automate their production processes as much as the technology would allow. In this review, a historical overview of the Dutch experiences is provided in the context of the international developments.
This study, conducted under the auspices of ANDEM, was aimed at determining the reasons for the inefficacy of platelet transfusions for thrombocytopenia of central origin, and to establish recommendations for therapy and prevention. It was based on a critical review of the literature. Three main causes of inefficacy were identified : an immunological conflict (mainly anti-HLA alloimmunization), clinical status, and the quality of platelet preparations (especially storage time). Our recommendations depend on several factors, such as the degree of urgency, the cause of platelet refractoriness, clinical status and the availability of platelet preparations. The mainstay of the recommendations is prevention, based on the quality of platelet preparations (as fresh as possible, in adequate number preferably obtained by apheresis and ABO compatible) and on the prevention of anti-HLA alloimmunization.
. 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.
Background: Buffy coats (BCs) are used as an alternative to platelet-rich plasma in the preparation of platelet concentrates (PCs). For this purpose the BCs have to be stored for some time at 20–24°C which implies cellular metabolic activity. However, little information is available concerning the effects of a number of factors which may influence the suitability of the preparation as the source of PC. Study design and methods:We studied the effects on BCs of a high and low gas permeability of the wall of the plastic containers, PL2209 and PL146, respectively, mixing versus non-mixing during storage for 48 h at 22°C, and two types of anticoagulant solutions, CPD and half strength citrate CPD (0.5CPD). The buffy coats were prepared by the bottom and top technique. The median values of volume and haematrocrit were 58–64 ml and 39–45%, respectively. A total of 48 BCs were tested. Blood gases, pH, bicarbonate concentration and haemolysis were determined in the blood mixtures and β-thromboglobulin (β-TG), lactate dehydrogenase (LDH), complement factor 3a, and elastase in the extracellular fluid. Results: The pH decreased in all units but to a lesser extent in PL2209 containers than in PL146 units. In the former the pCO2 decreased slowly in contrast to the latter where it increased by about 50%. Mixing during storage increased the pH and decreased the pCO2 in 0.5CPD-PL146 and CPD-PL2209 units, as compared to resting, while no effects of mixing were observed in the other groups. The pO2 decreased to low levels in PL146 units. The haemolysis and LDH release were higher in mixed than in unmixed units. The initial β-TG levels were lowest in 0.5CPD-PL146 units which also had the lowest 24-hour levels. The release of β-TG during storage was smallest in CPD resting units. The elastase release was significantly higher in 0.5CPD than in CPD units already from the beginning of storage and increased during storage at about the same rate irrespective of mixing. The C3a levels were higher in 0.5CPD-PL2209 units than in the other units at 2 h. Storage for 24 h caused an increase by 2–3 times of the original level without any clear relation to storage conditions. Conclusions: In BC units accumulation of CO2 occurs in containers with low gas permeability. These also show the most rapid pH decrease during storage. Prolonged holding of BCs puts extra emphasis on the need of satisfactory gas permeability of the container for platelet storage in BC-derived PCs. Continuous mixing causes red cell damage and does not seem to have any clear benefit. The release of granulocyte elastase was higher in 0.5CPD than in CPD units but there was no indication of an associated increase in platelet activation. Summary: Study of buffy coats stored in various media and containers at 22°C suggests that it is better to restrict storage to 24 h or less to avoid activation or other deleterious effects on the platelets.
BACKGROUND: Platelet concentrates (PCs) may be subjected to temperatures outside 20 to 22 degrees C during shipping or storage, which may have an adverse effect on platelet quality.
STUDY DESIGN AND METHODS: These studies systematically evaluated the effect of short- term exposure (≤ 24 hours) of platelets to temperatures above 22° or below 20° C as part of standard 5-day PC storage at 22° C, as well as the effect of long-term storage (5 days) at 24 and 26° C. For the short-term exposure studies, up to 6 units of Day 1 standard PCs were mixed, split, and returned to the containers. Test units were then stored without agitation in an incubator at a specific temperature (4, 12, 16, or 18° C) for various times up to 24 hours, after which they were stored with agitation at 22° C. One unit acted as control and was stored at 20 to 22° C throughout the 5-day storage period. Loss of platelet discoid shape was determined photometrically by the extent of shape change assay, by an increase in apparent platelet size by morphologic evaluation, and by swirling.
RESULTS: A gradual loss of platelet discoid shape occurred at temperatures below 20° C. For similar periods, a greater difference between test and control PCs was observed in units held at 4° C than in those held at 16° C. The data were fitted to an equation to relate platelet discoid shape (% of control) to exposure temperature and time. Assuming that a 20-percent decrease or more in the extent of shape change assay represents a significant loss in platelet viability, the equation predicts that such a loss occurs when the platelets are exposed to 16° C for ≥16 hours, to 12° C for ≥10 hours, or to 4°C for ≥6 hours, whereas exposure to 18° C for ≤24 hours has no significant effect. Storage for 5 days at temperatures ≤26° C was not associated with any significant reduction in platelet discoid shape or other measures of platelet quality.
CONCLUSION: There was a gradual loss of platelet discoid shape at exposure temperatures < 20°C, which worsened as temperatures decreased and exposure times increased to 24 hours. This relationship can be described in an equation that could be used as a guideline for allowable exposure conditions.
The possible beneficial role of white cells (WBCs) in donor blood has been investigated with respect to their capacity to remove bacteria. Preparations of buffy coat and whole blood, containing as well as reduced of WBCs, were inoculated with Staphylococcus epidermidis, S. aureus, Escherichia coli, Pseudomonas aeruginosa, and Propionibacterium species. Upon storage at room temperature, the presence of WBCs resulted in a reduction of the bacterial content. Units inoculated with S. epidermidis and E. coli were completely cleared of bacteria within 5 to 24 hours. On the other hand, S. aureus, after an initial reduction in number, started to multiply. In WBC-reduced units, the initial bacterial content remained unchanged for 5 hours, but the bacteria then exhibited vigorous growth within 48 hours in buffy coat and slower growth in whole blood. Propionibacterium sp. did not grow with or without WBCs. P. aeruginosa did not grow in buffy coat but showed a growth pattern similar to that of S. aureus in whole blood. The presence of WBCs in the donor blood during the first hours after collection thus seems to rid the blood of at least some species of bacteria. These results indicate that it would be favorable not to perform WBC reduction during blood collection and that several hours of contact can be needed to obtain sterility.
Platelet concentrates (PC) continues to be the only resource available to treat patients with qualitative and/or quantitative platelet defects. Currently we have three methods to prepare PC: platelet rich plasma (PRP), buffy coat (BC), and apheresis. In recent years, the characteristics of the products have improved significantly: greater yield of better platelets with less RBC and leucocyte content. At the present, BC and apheresis derived PC offer some advantages in comparison to PRP, as it can been prepared in additive solutions, culture them using sensitive methods or apply pathogen reduction technologies.
The use of platelet transfusions has risen considerably in the last years. Changes occur in platelet biochemical and membrane properties during storage. We have analyzed the effect of platelet preparation and storage of platelet function through the evaluation of platelet cytoskeletal reorganization.
A blood sample was obtained from the donor and platelets were separated as standard platelet-rich plasma (PRP) (120 g, 20 min) (PRE sample). Aliquots were also collected immediately after preparation using buffy coat procedure of platelet concentrates (day 0) and after 1, 3 and 5 days of storage. Cytoskeleton composition in both low- and high-speed cytoskeletal fractions of detergent-lysed platelets was analyzed by gradient SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Presence of each contractile protein was quantified by densitometry.
The method used to prepare platelet concentrates induced actin polymerization (actin increased to 163.5 +/- 4.8%, mean +/- SEM, n = 8, p < 0.001, considering actin values in PRE sample as 100%) with a concurrent increase in the association of actin-binding protein (ABP), myosin and alpha-actinin to the low-speed cytoskeletal fraction. During the first 24 hours of storage, cytoskeletal assembly was partially reversed (134.8 +/- 2.6% of actin, p < 0.001) and actin polymerization increased gradually to 144.3 +/- 5.8% and 153.2 +/- 5.1% at days 3 and 5, respectively (p < 0.001 for both days). ABP, myosin and alpha-actinin showed similar tendencies to those referred for actin. Conversely, during platelet preparation and storage, the contractile proteins associated with the high-speed cytoskeletal fraction decreased, due to reorganization of the contractile proteins to the low speed fraction.
The method used to prepare platelet concentrates (buffy coat procedure) induced cytoskeletal polymerization. This activating effect was partially reversed after 1 day of storage, although it increased progressively after 3 days of storage. The storage lesion may lead to defective cytoskeletal assembly in response to further stimulus. Analysis of cytoskeletal assembly is a sensitive method for detecting platelet activation caused by the concentrate preparation method and the storage conditions.
A recent review concluded that there was inadequate evidence to show a difference between buffy coat (BC) and platelet (PLT)-rich plasma (PRP) PLT concentrates prepared from whole blood. We hypothesized that 7-day-stored BC-PLTs would have superior autologous recoveries and survivals compared to PRP-PLTs and that both would meet the Food and Drug Administration (FDA) criteria for poststorage viability.
This was a randomized, crossover study design in healthy subjects who provided informed consent. Each participant donated a unit of whole blood on two occasions. In random order, either BC-PLTs or PC-PLTs were prepared after a 20 ± 2 °C overnight hold of the whole blood. PLTs were stored under standard conditions. On Day 7, fresh PLTs were prepared from 43 mL of autologous whole blood. The fresh PLTs paired with either BC-PLTs or PRP-PLTs were alternately labeled with (111) In or (51) Cr and simultaneously reinfused to determine recoveries and survivals. In vitro assays were performed on Days 1 and 7.
Fourteen subjects completed the study at two sites. No differences in poststorage PLT viabilities were observed between BC-PLTs and PRP-PLTs; recovery differences averaged 3.7 ± 2.4% (± SE, p = 0.15) and survival differences averaged 0.48 ± 0.56 days (p = 0.41). Neither type of PLTs met the current FDA criteria for either poststorage PLT recoveries or survivals.
We were unable to demonstrate that single-unit BC-PLTs stored for 7 days have superior poststorage viability compared to PRP-PLTs. Failure to meet the minimum FDA criteria for poststorage PLT viability raises questions regarding the acceptance thresholds of these metrics.
Two techniques for the preparation of platelet concentrate (PC), the standard platelet-rich plasma (PRP) and buffy coat (BC) methods, were compared in nine paired studies with regard to platelet harvest, white cell (WBC) contamination, and PC quality after 5 days of 22 degrees C storage. Platelet harvest using the BC method averaged approximately 56 percent of the whole blood level (6.2 x 10(10)/concentrate), which was less than the 76 percent achieved with the PRP-PC method (8.7 x 10(10)/concentrate). An additional 5 units collected into an experimental siphon bag for BC-PC processing showed improved platelet harvest (6.7 x 10(10)/concentrate, or approx. 70% of whole blood). WBCs remaining in the BC-PC averaged 0.19 x 10(8) per unit compared to 3.6 x 10(8) per unit for PRP-PC. Buffy coat processing produced red cell (RBC) units with 50 percent of the WBC contamination of conventionally prepared units (9.8 +/- 6.2 x 10(8)/unit vs. 18.9 +/- 7.1 x 10(8)/unit). The siphon bag further reduced WBC levels in the AS-3 RBC units (6.4 +/- 3.7 x 10(8)/unit). In vitro studies performed on Days 1 and 5 after collection showed no significant differences in platelet metabolic and biologic function or cell integrity. Beta-thromboglobulin and surface glycoprotein levels, indicators of platelet activation and membrane alteration, respectively, did not differ significantly in the PRP-PC and BC-PC; nor was lactate production higher in PRP-PC, despite the substantially higher WBC counts. Autologous in vivo platelet viability determinations were performed by using concurrent transfusion of 111In-labeled freshly drawn platelets and 51Cr-labeled stored platelets.(ABSTRACT TRUNCATED AT 250 WORDS)
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 +/- 52 x 10(9) platelets (mean +/- SD, range 203-490, n = 134), obtained from 4 BC pools and lower leukocyte contamination than before, 18 +/- 17 X 10(6) per preparation (range 1-73, microscopic counting, n = 38). The storage medium consisted of a mixture of plasma and PAS.(ABSTRACT TRUNCATED AT 250 WORDS)
The effect of rapid cooling to 20-24 degrees C of whole blood immediately after collection, using 'cooling units' with butane-1,4-diol and prolonged storage up to 24 h at ambient temperature was investigated in the whole blood and the subsequently prepared plasma, buffy coat and buffy-coat-poor red cell concentrate (BC-poor RCC) in saline-adenine-glucose-mannitol (SAG M) solution. Factor VIII:C content of the plasma (n = 10), after 24 h storage was 80 +/- 3% of the initial value. In routine procedures factor VIII:C content in the plasma (n = 129 pools of 20 donor units plasma) was 0.77 +/- 0.078 IU/ml, after storage of the whole blood for 16-20 h. In whole blood (n = 10), the 2,3-diphosphoglycerate (2,3-DPG) content of the red cells decreased from 4.36 +/- 0.55 to 1.47 +/- 0.6 mumol/ml red cells after 24 h storage at 20-24 degrees C. After storage of the BC-poor RCC (n = 10) at 2-6 degrees C for 1 week, the 2,3-DPG had dropped to 0.76 +/- 0.46 mumol/ml red cells. During the first 24 h of storage of whole blood, the adenine triphosphate (ATP) levels of the red cells remained stable. A mean increase of 20% of the initial value was observed after addition of SAG M solution. In the BC-poor RCC the ATP slowly decreased to 81 +/- 5% after 5 weeks and to 68 +/- 6.6% of the initial value after 6 weeks storage.(ABSTRACT TRUNCATED AT 250 WORDS)
Platelet concentrates (PCs) were prepared from single buffy coats derived from fresh blood and from blood units stored overnight, as well as from buffy coats that were stored overnight. The platelet yield from overnight-stored buffy coats was similar to that of fresh blood or overnight-stored blood. PCs were stored at 20-24 degrees C and on day 5 of storage, platelet aggregation with ADP was tested both at 37 and 25 degrees C. Stored platelets aggregated better at 25 degrees C than 37 degrees C. The maximal, aggregation (10 microM ADP) of stored platelets from overnight-stored buffy coats was 46 +/- 23% (n = 30), while that of stored platelets prepared either from fresh or overnight-stored blood was 27 +/- 21% (n = 29) and 22 +/- 15% (n = 29), respectively. Extracellular lactate dehydrogenase and ammonia levels, as well as elastase activity were similar in stored PCs of different origin. Our conclusion is that PCs prepared from overnight-stored buffy coat might also be suitable for storage and clinical use. In vivo studies are needed to confirm our findings.
We evaluated in vitro platelet function of platelet concentrates stored at 22 C for 5 days prepared either by the conventional pelleting procedure or platelet concentrates prepared from buffy coats by utilizing a novel bucket designed to support a suspended bag. For platelet concentrates from buffy coat, whole blood was centrifuged at 3,000 x g for 13 min, with all but 30cc of the cell poor plasma transferred to a satellite bag, followed by a second centrifugation at 170 x g for 5 min utilizing our novel centrifugation device. For pelleted platelets, whole blood was centrifuged at 2,000 x g for 3 min, platelet rich plasma removed, centrifuged, and the pellet resuspended in plasma. Leukocyte contamination in buffy coat platelet concentrates was reduced by 95% (p < 0.001) in comparison to pelleted platelets. Further, platelets from buffy coat platelet concentrates demonstrated significantly enhanced ADP-induced aggregation, increased recovery from hypotonic shock, higher morphology scores, and reduced GMP-140 expression in comparison to pelleted preparations. No differences in O2 consumption, CO2 production, pH and total ATP were observed between the two types of preparations at day 5 of storage. Our results indicate that platelet concentrates from buffy coat, prepared by a suspended storage bag centrifugation technique, are superior with respect to in vitro platelet function when compared to pelleted platelets.
The importance of white cell (WBC) reduction in platelet concentrates (PCs) for component quality is undetermined.
Eleven paired components, each derived from one of two whole-blood units given by a single donor on the same day, were studied. One PC was WBC reduced by filtration with an in-line, integral, prototype filter, and the other was produced from unfiltered platelet-rich plasma (PRP) by a standard method. In vitro tests performed on Day 1 and Day 5 were blood gases, plasma lactate, glucose, platelet ATP, mean platelet volume, morphology score, hypotonic stress ratio, extent of shape change in response to ADP, and beta-thromboglobulin. After 5 days of storage, each component pair was labeled with 51Cr or 111In and transfused for the estimation of percent recovery and survival.
PCs using the in-line, prototypic filter had a platelet loss of approximately 15 percent and a variable 1 to 3 log10 reduction (average, 95%) in WBC content. The variation in filter WBC removal was related to PRP WBC content and indicated that the filter did not have the capacity for a 3 to 4 log10 removal when PRP WBC content exceeded 1 x 10(8). The in vitro and in vivo measures of platelet quality showed no meaningful differences between filtered and unfiltered PCs by paired t test. The mean differences in posttransfusion percent recoveries and survivals were 0.9 +/- 2.9 percent and 4 +/- 13 hours, respectively. Additional studies were performed using a larger filter with improved capacity. Those studies (n = 18) showed a significant improvement in filtration time and platelet yield and a consistent 3 to 4 log10 reduction in WBCs. Filtration time was 6.6 +/- 2.7 minutes, total PC WBCs were 9.6 +/- 4.6 x 10(4), and total PC platelets were 7.8 +/- 1.8 x 10(10) (mean +/- SD).
Prestorage filtration of PRP and the preparation of filtered platelets do not result in any significant beneficial or adverse effect on subsequent platelet quality. With the large-capacity filter, consistent WBC reduction and good platelet yields are achieved.
The effect of prestorage filtration on the quality of apheresis platelet concentrates stored for transfusion is undetermined.
Investigation of 11 plateletpheresis components used a concurrent paired-study design. On the day of collection, each component was equally divided into two suspensions; one half was filtered, and the other half was not. Each suspension was stored for 5 days. In vitro testing was performed on the day of collection (Day 0) for cell counts and on Day 5 for measurements of lactate, glucose, blood gases, pH, platelet ATP, hypotonic stress ratio, extent of shape change in response to ADP, tissue necrosis factor alpha, interleukin 8, interleukin 1 alpha, interleukin 1 beta, interleukin 6, and platelet surface glycoproteins by flow cytometry. At the end of the 5-day period, a sample was taken from each of the two suspensions, radiolabeled with either 51Cr or 111In, and transfused concurrently. Posttransfusion samples were drawn for measurements of recovery and platelet survival and for functional assessment of the ex vivo ability of the circulating radiolabeled platelets to aggregate in response to ADP.
The apheresis component had a mean platelet yield of 3.2 +/- 0.4 x 10(11) and a white cell yield ranging from 1 x 10(5) to 1 x 10(8), with a median of 2 x 10(7). Filtration resulted in a platelet loss of approximately 10 percent and a variable 2 to 3 log10 reduction in white cell content. No significant differences between filtered and unfiltered suspensions in paired t tests that would likely have an impact on platelet quality were observed in the in vitro tests. The in vivo recovery and survival were highly similar and not statistically different in filtered and unfiltered paired suspensions: the mean difference was 1.2 +/- 4.0 percent for recovery and 7.0 +/- 15 hours for survival. The functional assessment by aggregation to ADP showed no difference between filtered and unfiltered suspensions. A small decrease in tumor necrosis factor alpha and interleukin 8 was evident in the filtered suspension as compared to levels in the unfiltered suspensions.
Prestorage white cell reduction in apheresis components resulted in WBC reduction by several log10 with no evident adverse effect on platelet viability or function.
Leukocyte-depleted platelet concentrates were prepared from pools of 4 buffy coats on the day after blood collection (BC-PC). The storage medium was composed of citrate phosphate dextrose plasma and a platelet-additive solution. Autologous transfusions of 111In-labelled platelets in 9 healthy volunteers were performed on the day of preparation (day 1) and on day 5. The recovery was 54.6 +/- 8.7 (day 1) and 51.9 +/- 10.4% (day 5), T1/2 was 101 +/- 28 and 61 +/- 9 h, respectively. The survival was 8.3 +/- 1.7 and 5.7 +/- 1.0 days, respectively, using linear plot, and 7.8 +/- 2.0 and 5.8 +/- 0.5 days using the multiple hit method. In a prospective clinical study a comparison of the corrected posttransfusion increments was made between BC-PCs and apheresis-PC, and between fresh (1-2 days) and stored (3-5 days) preparations. No difference was found between BC-PCs and apheresis PCs. However, fresh BC-PCs gave higher increments than stored BC-PCs. A slight numerical difference between fresh and stored apheresis-PCs was not statistically significant. It is concluded that the BC-PC method results in platelets of equal quality to apheresis-PC.
Random-donor platelet concentrates (PC) prepared from pooled buffy coats have recently been described as an alternative method for platelet preparation. We evaluated such PCs in the clinical setting compared with a standard PC from platelet apheresis. PCs were prepared either from pools of buffy coats (BC-PC) or from single donors (SD-PC) with the cell separator CS-3000 plus. PCs were stored for up to 5 days before transfusion. We compared fresh PC (day 1) with stored (day 2-3) and long-stored PC (day 4-5). For analysis, platelet increment in the recipient was determined immediately and 16-22 h (mean 20 h) after transfusion, corrected for total body area and transfused platelets (CCI). A total of 316 PCs were administered to 36 thrombocytopenic patients suffering from various hematological disorders. Patients with detectable HLA or platelet-specific antibodies or splenomegaly were excluded from the study. Mean platelet content of the PC was 262 x 10(9) for BC-PC and 251 x 10(9) for SD-PC. The 20-h CCI after transfusion of fresh PC was slightly higher with BC-PC than with SD-PC (14.5 versus 11.9; p = 0.19), but values did not differ significantly between the two types of PC on any day of storage. For BC-PC, 20-h CCI decreased with further storage by 30% (10.2; p = 0.02). For SD-PC a decrease by 9% was not significant. In conclusion, platelet concentrates prepared from pools of buffy coats showed excellent transfusion results when administered fresh, but storage decreased the CCI by 30%. No significant difference from PCs from plateletpheresis was observed on any day of storage. Both types of platelet concentrates were capable of sufficient platelet increment even when stored for up to 5 days.
The functional capacity of transfused platelets was evaluated with in vitro bleeding time (IVBT) and corrected count increment (CCI) in order to compare platelet concentrates (PCs) derived from pooled buffy coats (BC-PCs) with PCs collected by apheresis (A-PCs). The suspension medium in the BC-PCs was 30% CPD plasma and 70% of an additive solution (containing sodium and potassium chloride, sodium citrate and phosphate, mannitol), and in the A-PCs the medium was 100% CPD plasma. IVBT was evaluated using a Thrombostat 4000/2. BC-PC and A-PC were transfused 57 and 41 times, respectively to 36 patients with chemotherapy-induced thrombocytopenia. PCs transfused within 2 days of donation were considered fresh, and those transfused within 3-5 days were considered stored. IVBT was determined before, as well as 10-30 min and 24 h after transfusion; CCI was determined 10-30 min and 24 h after transfusion. The median pretransfusion IVBT value was 486 s. It was measurable in 21 of 98 (21%) of the transfusions, i.e. below the cutoff limit of 486 s. Ten to 30 min after transfusion, the IVBT showed a measurable reduction in 90% of the transfusions with fresh BC-PCs, 92% of those with fresh a-PCs, 63% of those with stored BC-PCs and 79% of those with stored A-PCs. After 24 h, the corresponding values were 63% for fresh BC-PCs, 50% for fresh A-PCs, 26% for stored BC-PCs and 38% for stored A-PCs. The median value of CCI 10-30 min after transfusion was 20 for fresh BC-PCs, 17 for fresh A-PCs, 16 for stored BC-PCs and 14 for stored A-PCs. The difference in IVBT between fresh and stored BC-PCs was significant (p = 0.032), unlike that between fresh and stored A-PC. After 24 h the corresponding values were 7 for fresh BC-PCs, 4 for fresh A-PCs, 4 for stored BC-PCs and 3 for stored A-PCs. When all transfusions with fresh PCs (BC-PCs + A-PCs) were compared with all transfusions with stored PCs, a statistical difference was demonstrated in both CCI (p = 0.027) and IVBT (p = 0.043). Spearman's rank correlation coefficient (rs) was -0.41 between CCI and IVBT < 486 s 10-30 min after transfusion, and -0.55 between the posttransfusion platelet count and IVBT, indicating a relatively poor correlation between CCI and IVBT, and a slightly better correlation between platelet count and IVBT. In conclusion, BC-PCs showed a slightly higher CCI and a better response in IVBT than A-PCs. No statistical difference was demonstrated between BC-PCs and A-PCs transfused within 2 days after donation, with respect to function and recovery in vivo. BC-PCs stored for 3 days or more showed about the same CCI and IVBT as stored A-PC but significantly lower CCI and higher IVBT than fresh BC-PCs. This may indicate that the preparation and/or storage conditions were not optimal. IVBT seems to be a useful possibility to test the in vivo behavior of transfused platelets.
The aim of this study was to compare the clinical effect of transfusion of platelet concentrates (PC) prepared from pooled buffy coats (BC) and PCs collected from a single donor (SD) by an apheresis technique. The influence of storage time and various clinical conditions was also studied. Thirty-two patients suffering from haematological malignancies were given a total of 326 platelet concentrates; 180 BC-PCs and 146 SD-PCs, median 7 transfusions per patient. BC-PCs contained 312 +/- 52 x 10(9) and SD-PCs 383 +/- 133 x 10(9) platelets/unit (mean +/- SD). The mean storage time of BC-PC was 3 d and that of SD-PC 1 d. The mean platelet count of the patients before transfusion was 11 +/- 8 x 10(9)/L. Regression analysis showed a significant decrease of the post-transfusion platelet corrected count increment (CCI) during storage of PCs for 1-5 d (BC-PC: p < 0.01; SD-PC: p < 0.05). There was no difference in platelet increment between BC-PC and SD-PC. Human leukocyte antigen (HLA) alloimmunization was the major cause of clinical refractoriness to random donor platelet transfusions but splenomegaly also caused low CCI values.
Whole blood-derived buffy coat (BC) has become an alternative source from which to prepare random-donor platelet concentrates. The influence of prolonged storage of BC prior to platelet concentrate preparation is a matter of controversy. The impact of BC storage on cytokine release was evaluated and the platelet activation quantified.
BCs were prepared from whole-blood donations after hard-spin centrifugation. After 1, 3, 6, 12, and 24 hours of storage at 22 degrees C without agitation, samples were withdrawn for cell count and blood gas analysis and measurement of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor alpha and platelet factor 4. Platelet surface markers CD41a, CD42b, CD62P, and CD63 were analyzed by flow cytometry, and the antibody-binding sites were quantified by using microbeads.
Inflammatory cytokines IL-1beta, IL-6, and tumor necrosis factor alpha were hardly detectable in stored BCs but levels of IL-8 increased in 25 percent of BCs after 24 hours. A constant increase in platelet factor 4 was observed, which accelerated after 12 hours of storage. Analysis of platelet surface markers showed an initial decrease of platelet activation, followed by an increase after storage for 12 to 24 hours.
Storage of BCs for up to 12 hours without agitation showed a good preservation of platelets but storage of BCs for 24 hours resulted in increased platelet activation and significantly higher release of platelet factor 4 and IL-8. Stored BCs might well be suitable for platelet preparation, but their storage time should not greatly exceed 12 hours.
Buffy coats (BCs) are used as an alternative to platelet-rich plasma in the preparation of platelet concentrates (PCs). For this purpose the BCs have to be stored for same time at 20-24 degrees C which implies cellular metabolic activity. However, little information is available concerning the effects of a number of factors which may influence the suitability of the preparation as the source of PC.
We studied the effects on BCs of a high and low gas permeability of the wall of the plastic containers, PL2209 and PL146, respectively, mixing versus non-mixing during storage for 48 h at 22 degrees C, and two types of anticoagulant solutions, CPD and half strength citrate CPD (0.5CPD). The buffy coats were prepared by the bottom and top technique. The median values of volume and haematrocrit were 58-64 ml and 39-45%, respectively. A total of 48 BCs were tested. Blood gases, pH, bicarbonate concentration and haemolysis were determined in the blood mixtures and beta-thromboglobulin (beta-TG), lactate dehydrogenase (LDH), complement factor 3a, and elastase in the extracellular fluid.
The pH decreased in all units but to a lesser extent in PL2209 containers than in PL146 units. In the former the pCO2 decreased slowly in contrast to the latter where it increased by about 50%. Mixing during storage increased the pH and decreased the pCO2 in 0.5CPD-PL146 and CPD-PL2209 units, as compared to resting, while no effects of mixing were observed in the other groups. The pO2 decreased to low levels in PL146 units. The haemolysis and LDH release were higher in mixed than in unmixed units. The initial beta-TG levels were lowest in 0.5CPD-PL146 units which also had the lowest 24-hour levels. The release of beta-TG during storage was smallest in CPD resting units. The elastase release was significantly higher in 0.5CPD than in CPD units already from the beginning of storage and increased during storage at about the same rate irrespective of mixing. The C3a levels were higher in 0.5CPD-PL2209 units than in the other units at 2 h. Storage for 24 h caused an increase by 2-3 times of the original level without any clear relation to storage conditions.
In BC units accumulation of CO2 occurs in containers with low gas permeability. These also show the most rapid pH decrease during storage. Prolonged holding of BCs puts extra emphasis on the need of satisfactory gas permeability of the container for platelet storage in BC-derived PCs. Continuous mixing causes red cell damage and does not seem to have any clear benefit. The release of granulocyte elastase was higher in 0.5CPD than in CPD units but there was no indication of an associated increase in platelet activation.
Study of buffy coats stored in various media and containers at 22 degrees C suggests that it is better to restrict storage to 24 h or less to avoid activation or other deleterious effects on the platelets.
The percentage of platelets that retain discoid form correlate with their post-transfusion viability and therefore they may act as a simple indicator for the quality of the stored platelet concentrates (PCs). The morphological score (MS) of platelets, given by Kunicki, requires the index of different forms of platelets: discoid, spherical, starlike and bizarre. In the present study, we intended to clarify whether the morphological score of discoid platelets alone could serve as a method for the estimation of PCs quality. During the first five days after preparation, samples of standard platelet concentrates (SPCs) and of leucodepleted platelet concentrates (LDPCs) were analyzed morphologically by immersion light microscopy using smears stained by the May-Grünwald-Giemsa method. In addition to the MS, an original Modified Morphological Score (MMS), for a population of 200 platelets, was used to count platelets with the discoid shape. The mean MMS ranged (first to fifth day) from 192.6 to 166.6 with SPCs and from 197.2 to 185.4 with LDPCs. There is a significant difference (ANOVA) between: (a) PCs prepared by different techniques (p < 0.01); (b) the quality of the same PCs during the storage (p < 0.0001).
Purpose: Prestorage leukocyte filtration of platelet concentrates (PC) has been considered to reduce the incidence of transfusion reactions. It is a current matter of debate whether prestorage leukocyte filtration has an impact on the storability of platelets and on the transfusion results of PC. Methods: We investigated the transfusion results of PC from pooled buffy-coats in hematological patients without known refractoriness to platelets and compared bedside filtration (n=228) versus prestorage filtration (n=271) with an Auto-Stop BC filter (Pall, Dreieich, Germany). The following parameters were determined: leukocyte and platelet content of the PC, duration of storage, platelet count of the patient before transfusion, 1 hour and 24 hours after transfusion, platelet increment and corrected count increment (CCI) 1 hour and 24 hours after transfusion. Results: The leukocyte content of the bedside filtered group was 66 ± 50 ×106 while it was <0.1×106 for the prestorage filtered group. Platelet counts before transfusion (13.7/nl vs. 16.6/nl), platelet content of the PC (2.6×1011 vs. 2.7×1011) and the duration of storage (2.7 vs. 2.6 days) were almost identical in both groups. There was no significant difference for the platelet increment after 24 hours (14.6/nl vs. 14.9/nl) or the CCI after 24 hours (12.0/nl vs. 11.1/nl). Conclusions: Prestorage leukocyte reduction of PC had no impact on the CCI. When the leukocyte content of PC was <1×108, prestorage leukocyte reduction had no beneficial effects on the transfusion results.
Cryopreservation of platelets is of great interest, since it could extend the shelf life of therapeutic platelet concentrates and facilitate stockpiling and inventory control in blood banking. Despite the use of many cryopreservation procedures the optimal cryopreservation procedure is not defined yet. We have compared the cryopreservation of human platelets by various protocols employing controlled-rate and non-controlled-rate freezing procedures in combination with different concentrations of DMSO (6% and 10%) or 5% DMSO + 6% HES combination. After storage for 1 to 3 months, samples were thawed and analyzed. Measurements included cell recovery, platelet viability according to hypotonic shock response (HSR), platelet aggregation with ADP, morphological and ultrastructural properties of defrozen platelets. Our findings show that the application of our original procedure for controlled-rate freezing consisting of six cooling steps (cooling rate 1 degree C/min) with compensation of released heat of fusion (cooling rate 2 degrees C/min) has significantly influenced the quality of thawed platelets. At the same time, a concentration of 6% DMSO proved to be the most effective. In summary, cryopreservation of human platelets using controlled-rate freezing procedure in combination with lower (6%) DMSO concentration resulted in less damage from freezing and higher recovered function of platelets.
Filtered PLT concentrates (PCs) were prepared in plasma pooling three (for children) or six buffy coats (BCs; for adults) after holding them a maximum of 4 hours (blood bags collected in the afternoon) or 18 hours (blood bags collected in the morning).
With flow cytometry, PCs prepared after holding BCs 4 or 18 hours were compared. BCs removed from whole-blood donations in quadruple bag packs ("top-top") were held 4 or 18 hours before pooling them with a sterile connecting device. After the BCs were centrifuged, the supernatant was transferred through a BC filter (Autostop, Pall Medical) to a CLX bag. Samples for analysis were collected from the whole-blood bag, BCs, and PCs immediately after preparation and after 1, 3, 5, and 7 days of storage on a flat-bed agitator at 22 +/- 2 degrees C. The main PLT membrane glycoproteins (GPs, IIb-IIIa, IV, and Ibalpha), some of their ligands (fibrinogen, fibronectin, and VWF), activation-dependent antigens (CD62P and CD63), and procoagulant activity markers (annexin V and bound coagulation FV-Va) have been studied.
In the 12 PCs (six pools of 3 units each group) studied, a minor increase in activation markers during preparation was observed. During the storage, a significant increase in the expression of GPIIb-IIIa, CD62P, CD63, annexin V, and FVa was measured. After 5 days of storage, only the percentage of PLTs with bound fibrinogen was significantly greater in PCs prepared after holding BCs for 4 hours.
In PCs prepared after holding BCs 4 or 18 hours before pooling and filtering, only a minor significant difference in the percentage of PLTs with bound fibrinogen was found after 5 days of storage. This difference is probably of little, if any, transfusional significance.
. To study the influence of contaminating leukocytes on the storage conditions of platelet concentrates (PC), various amounts of leukocytes were added to identical PC. From 12 blood donations, 12 leukocyte-poor PC were prepared and pooled. Subsequently, the pool was divided into 12 identical PC. The plasma volume of the PC was 58.6 ± 0.6 ml, the platelet concentration was 1.01 ± 0.04times 109/ml (mean ± SD) and the red cell contamination did not exceed 107 per PC. To 4 groups of 3 PC, pooled leukocytes were added from the same 12 blood donations. The leukocyte contamination for each group of 3 PC was 0.14 ± 0.05, 1.96 ± 0.09, 5.53 ± 0.98 and 13.0 ± 0.93 times 106/ml (mean ± SD) for groups I-IV, respectively. The PC were stored for 7 days at 22 °C in normal polyvinylchloride bags. A significant correlation was found between increasing concentrations of leukocytes in the PC and the drop in pH (r=-0.93), glucose consumption (r=-0.91), lactic acid production (r=0.93) and release of lactate dehydrogenase (r=0.92) during storage of the PC. The excretion of β-thromboglobulin, depletion of platelet adenine nucleotides, decreased ability to incorporate 3H-adenosine into metabolic nucleotides and poor morphology of the platelets were also significantly correlated with an increased number of leukocytes in the PC. These data show that high concentrations of leukocytes in PC have a significant detrimental effect on the viability of platelets during storage at 22 °C. We conclude that for good storage conditions of PC, the upper limit of leukocytes per PC should not exceed 107.
. A special insert was developed for centrifuge cups in order to prepare leukocyte-poor platelet concentrates from buffy coats by using quadruple citrate phosphate dextrose-saline adenine glucose mannitol systems from different manufacturers. Each centrifuge cup could contain up to 4 sets of double bags allowing the preparation of 24 platelet concentrates per run. Optimal conditions for centrifugation of the buffy coats in the inserts were found to be 6 min at 380 g (2,150 g min). A platelet count of 69 ± 19 × 109 and a leukocyte contamination of 14± 10.5 × 106 per platelet concentrate was thereby obtained in a plasma volume of 63 ± 10.5 ml (mean ±SD). The method described allows large scale production of leukocyte-poor platelet concentrates from buffy coats in a closed system.
Six citrate phosphate dextrose (CPD)-saline adenine glucose mannitol (SAG M) quadruple systems were evaluated for the preparation and storage of leukocyte-poor platelet concentrates (PC) from buffy coats. The platelet storage bags examined were manufactured from normal polyvinylchloride (PVC) or special-type plastics. Biotest supplied PVC 76 (n = 14) and PVC 763 (n = 16) NPBI supplied PSV 3277 (n = 15) and DPL-110 (n = 14) and Terumo supplied Teruflex (n = 18) and molded Teruflex (n = 14). The PC were stored for 7 days at 22 degrees C on a horizontally shaking platform. Cell counts, pH, PO2, PCO2, morphology score and swirling patterns were monitored at 5, 72, 120 and 168 h. The plasma volumes averaged 63 ml and ranged from 39 to 81 ml, the overall mean +/- SD platelet concentration was 0.89 +/- 0.33 X 10(9)/ml. None of the PC had a leukoyte count higher than 10 X 10(6) per unit. After storage for 168 h, the pH ranged from 6.56 to 7.40 for all brands. The PO2 remained stable and even rose significantly (p less than 0.05) during storage in the NPBI PSV 3277 and Terumo molded Teruflex bags. The PCO2 decreased equally in all bags. Morphology scores were well maintained in 98% of all PC for up to 120 h, and in 83% at 168 h. A swirling pattern score of 2.5 or greater predicted with a specificity of 100% a good morphology score in the PC.(ABSTRACT TRUNCATED AT 250 WORDS)
A new blood container material (PL 1240 plastic) made of polyvinyl chloride containing a tri(2-ethylhexyl) trimellitate plasticizer was evaluated in three laboratories. When platelet concentrates (50-60 ml) were stored on a variety of agitators for 7 days at 22 +/- 2 degrees C, poststorage pH (mean +/- SD) ranged from 7.29 +/- 0.05 (6 rpm elliptical rotator) to 6.87 +/- 0.8 (70 cycles per minute flatbed agitator). The platelet counts ranged from 1.51 +/- 0.12 to 0.95 +/- 0.36 X 10(6) per microliter. Morphology scores and hypotonic shock response values of platelets stored 7 days in PL 1240 plastic containers were better than those noted following 3-day storage of control platelets in PL 146 plastic containers. The percent discharge of lactic dehydrogenase from platelets stored 7 days in PL 1240 plastic containers for 3 days (p less than 0.05). Mean platelet recoveries of 44 +/- 15 percent (n = 11; 111Indium) and 39 +/- 8 percent (n = 29; 51Chromium) were seen when autologous platelets were infused following 5-day storage in PL 1240 plastic bags. Platelet half-lives of 3.6 +/- 0.4 (n = 9) 4.1 +/- 0.4 (n = 20) days were reported in the two laboratories which used 51Cr labeling, while survival values of 7.0 +/- 1.0, 2.8 +/- 0.8, and 5.4 +/- 1.9 days were seen when data from the 111Indium studies (n = 11) were analyzed using linear, exponential, and multiple hit programs, respectively. Platelets stored for 5 days also were administered to 13 thrombocytopenic oncology patients.(ABSTRACT TRUNCATED AT 250 WORDS)
A special insert was developed for centrifuge cups in order to prepare leukocyte-poor platelet concentrates from buffy coats by using quadruple citrate phosphate dextrose-saline adenine glucose mannitol systems from different manufacturers. Each centrifuge cup could contain up to 4 sets of double bags allowing the preparation of 24 platelet concentrates per run. Optimal conditions for centrifugation of the buffy coats in the inserts were found to be 6 min at 380 g (2,150 g min). A platelet count of 69 +/- 19 X 10(9) and a leukocyte contamination of 14 +/- 10.5 X 10(6) per platelet concentrate was thereby obtained in a plasma volume of 63 +/- 10.5 ml (mean +/- SD). The method described allows large scale production of leukocyte-poor platelet concentrates from buffy coats in a closed system.
A method is described to prepare platelet concentrates from the buffycoat of citrate‐phosphate‐dextrose (CPD) blood in a closed four‐bag system and to store the platelets in autologous plasma under sterile conditions. After separation of the blood into plasma, buffycoat and leukocyte‐ and thrombocyte‐poor red cell concentrate, saline‐adenine‐glucose‐mannitol (SAGM) was added to the red cells. Next the platelets were concentrated in plasma by a second centrifugation and were transferred to the 600‐ml bag that previously contained the SAGM. The platelets were stored for 72 h at 22°C on a platform rotator at 1 cycle per second. The mean volume of the platelet concentrates (n = 12) was 61 ml, with an average platelet content of 72 times 10 ⁹ . The mean leukocyte contamination was only 17 times 10 ⁶ ; erythrocytes were not detected. The aggregation of the platelets with 1 μ M of ADP was normal.
After 72 h of storage, the mean pH was 7.1 ± 0.1. The PO 2 always remained above 17 mm Hg. No loss of thrombocytes had occurred during storage. The platelets had retained their discoid shape. The aggregation response to ADP had disappeared, however.
The in vivo viability of the platelets was determined in 8 healthy volunteers. Autologous platelets, stored for 72 h at 22°C, were labeled with ⁵¹ Cr and reinfused [43 (±9)% recovery at 15 min after reinfusion]. The mean survival time in vivo of the platelets was 6.8 (±0.7) days.
These experiments indicate that with the four‐bag SAGM system leukocyte‐poor platelet concentrates can be prepared which remain in optimal condition for at least 72 h. Moreover, a high amount of plasma is yielded and, with SAGM as additive, leukocyte‐ and platelet‐poor red cell concentrates can be stored for 5 weeks.
Plastic storage bags designed to optimize O2 and CO2 transfer to preserve platelets for 7 days prior to transfusion were studied in vivo and in vitro. Platelets stored 7 days in second-generation CLX bags were compared to platelets stored 3 days in standard (CL-3861) 3-day storage bags and platelets transfused within 24 hours of collection. The CLX bags maintained concentrate pH at a mean of 6.85 +/- 0.03 (SEM) after 7 days, while in standard bags after 3 days of storage, the mean pH was 6.46 +/- 0.03. A smaller proportion of platelets stored 7 days in CLX bags were discarded because of a pH less than 6.0 compared to those stored 3 days in CL-3861 bags (10 vs 21%). Poststorage pH showed strong correlation with concentrate platelet count and weak correlation with concentrate white cell count in both bag types. There was no significant difference in the mean corrected platelet count increments between platelets stored 7 days in second generation CLX bags and those stored 3 days in CL-3861 bags (10,000 and 12,200 at 1 hour, and 7000 and 7500 at 24 hours, respectively) following transfusion to 16 thrombocytopenic recipients. However, transfusion of fresh platelets achieved mean corrected increments at both 1 and 24 hours posttransfusion that were higher than seen with either group of stored platelets (20,100 at 1 hour and 10,800 at 24 hours). Platelets can be stored 7 days in second-generation CLX blood bags with results comparable to those of platelets stored 3 days in standard bags.
Recent studies have shown that the incidence of alloimmunization due to repeated platelet transfusions from random donors may be reduced by the use of leucocyte-poor blood components. These results were confirmed by this study, where 16% of patients with acute leukaemia undergoing initial chemotherapy and receiving leucocyte-poor blood components developed lymphocytotoxic antibodies, compared with 48% of patients in a control group receiving standard (non-leucocyte-depleted) blood components. In a third group, who received leucocyte-poor blood components and HLA-matched platelets, none of the patients developed lymphocytotoxic antibodies. There was a low incidence of platelet-specific antibodies (8%) but no difference between the three groups. Improved methods of removing leucocytes from blood components appear to offer the best approach for minimizing HLA alloimmunization, as the provision of HLA-matched platelet donors for prophylactic platelet support of all patients is not feasible.
A statistical method is described for determining which recipient factors influence the efficacy of platelet transfusions in the treatment of thrombocytopenia caused by bone marrow depression. The method, known as interaction effects detection, is useful because it will relate a dependent variable 12-hour posttransfusion in vivo platelet increment which is numerical to independent variables (recipient factors), which are not necessarily numerical or even rank ordered, in such a manner as to reveal the interactions among the independent variables. The results of the analysis suggest that seven of the 23 independent variables could be key ones in determining the efficacy of a transfusion, as measured by the dependent variable.
To determine the degree of damage occurring during preparation and storage of platelet concentrates, the percent release of B-thromboglobulin (BTG) and percent leakage of the cytosolic protein lactic dehydrogenase was determined sequentially from phlebotomy to the end of storage for 72 h at 20-24 degrees C. The effect of storage temperature, pH, and radiation was also evaluated. The results showed that during preparation of platelet concentrate a large degree of release was found after resuspension of the platelet button formed after the high-speed centrifugation. During storage the percent BTG release increased from 18.1 to 40.2% (p less than 0.05). The percent release seen during storage at 4 degrees C (72 h) was 19.2%, while that seen for platelets subjected to temperature cycling at 4-37 degrees C was 24.9%. Both of these values were significantly less (p less than 0.05) than that seen for concentrates stored at room temperature. A negative correlation between pH and BTG release was found (r = -0.64). Irradiation to 10,000 rad did not induce the release reaction or lactic dehydrogenase leakage. We conclude that the degree of in vitro platelet release is dependent on the preparative manipulations, and gentler protocols for preparation and storage of platelets should be investigated.
We studied the transfusion response from random donor platelet concentrates in 15 stable multitransfused, thrombocytopenic patients by comparing the platelet counts measured before and 20 hours after transfusion. The observed platelet increments were corrected (corrected increment, C.I.) for the number of units of platelet concentrate transfused and the patient's body surface area in square meters (platelets/microliter per unit/m2). Using platelet concentrates stored for less than 24 hours, the patients achieved a median C.I. of 9500 (range: 5000-18,000). When platelet concentrates stored for 24 to 48 hours or 48 to 72 hours were given, the median C.I. markedly decreased to 1000 (range: 0-4800) and 0 (range: 0-5100), respectively (p less than 0.001). These differences could not be explained by further recipient alloimmunization. Transfusion with platelet concentrates less than 24 hours old on a second occasion, bracketing the transfusions of older platelet concentrates, resulted in a median C.I. of 7200 (range: 5400-14,500). Similar results were obtained in three patients when HLA-identical sibling platelet concentrates were employed. In vitro tests, including pH, morphology, and aggregation, demonstrated no statistically significant differences among the platelet concentrates stored for less than 24 hours, 24 to 48 hours, and 48 to 72 hours. These studies suggest that, although platelet concentrates can be stored for 72 hours without loss of in vitro function, the in vivo recovery is significantly diminished after 24 hours of storage, and preferably patients should not be transfused prophylactically with platelet concentrates greater than 24 hours old.
From January 1972 to July 1974, 28 patients with bone marrow depression due to aplastic anemia or cytostatic treatment, were transfused with packed cells and platelet concentrates, both containing 10-20% of the amount of leukocytes present in whole blood. Of these patients, 26 (93%) became refractory to random platelets. Since July 1974, 68 patients have been given red cells filtered through cotton-wool, a procedure which removes over 97% of the leukocytes, and leukocyte-poor platelet suspensions obtained by an additional centrifugation step. Of this latter group, 16 patients (24%) became refractory. Fifty-two recipients were non-refractory to random platelet transfusions after an exposure time of at least 6 weeks and maximally 32 weeks. A possible explanation is that platelets are less immunogenic than leukocytes; on the other hand platelets may not be immunogenic at all with regard to induction of HLA antibodies, and the occurrence of the immunization is purely the result of the contamination with leukocytes in the red cell and platelet preparations.
Seventy-nine platelet transfusions to 73 thrombocytopenic patients with cancer were analyzed to determine whether a platelet count obtained one hour after transfusion could help differentiate between alloimmunization and other clinical factors that result in rapid platelet destruction. These transfusions were selected because 18- to 24-hour increments were inadequate in response to fresh, random donor platelets. A corrected count increment (Cl) (Cl=[posttransfusion count-pretransfusion count]Xbody surface area [sq m]/platelets transfusedX10'') at one hour of 10X103/microliter or greater was associated with absence of lymphocytotoxic antibody, whereas increments of less than 10X103/microliter were generally associated with high levels of strongly cytotoxic antibody. HLA-matched transfusions produced no improvement in increments when the previous one-hour Cl had been 10X103/microliter or greater, whereas in the other group significantly better increments were obtained. A one-hour posttransfusion count is a simple test that correlates well with the presence of antibody against HLA antigens, is valuable in predicting the need for HLA-matched platelets, and helps avoid wasteful, empirical use of such transfusions.
Platelet concentrates stored in plasma for 72 h at 22 ° prepared from buffy coats of CPD blood collected in a quadruple-bag SAG M system
- Pietersz
Platelet transfusion therapy. One‐hour posttransfusion increments are valuable in predicting the need for HLA‐matched preparations
- Daly P.A.