Reactions to platelet transfusion: the effect of the storage time of the concentrate

Random platelet concentrates were pooled and depleted of leucocytes by centrifugation immediately prior to transfusion. The incidence and severity of reactions to 570 leucocyte-poor platelet transfusions in 74 patients were studied. An overall transfusion reaction rate of 13.7% was observed. The reaction rate to platelets stored for less than 3 days (8.7%) was significantly different from the reaction rate to platelets stored for 3 days or more (17.6%). Minor reactions as well as moderate and severe reactions were more frequent in the latter group. As most of the white blood cells were removed prior to transfusion, it is suggested that the reactions result from the transfusion of pyrogenic and/or vasoactive substances accumulated in the plasma of the concentrate during storage.     

Extension of platelet concentrate storage

Extension of the storage time of platelet concentrates in a satellite bag which is part of a new blood bag system was studied by reinfusing autologous 51Cr-labeled platelets into normal volunteers, and measuring postinfusion platelet counts and bleeding times in patients requiring platelet transfusions. This satellite bag, made of polyvinylchloride plasticized with a new agent, was found to protect platelet concentrates against fall of pH better than other containers studied. This protection was felt to be due to the greater gas permeability of the new plastic. Mean in vivo recovery and half-life (greater than 31% and 3.3 days, respectively) of autologous reinfused platelets were satisfactory following 5 days of storage. Following 7 days of storage, mean recovery was 41 percent and half-life was 2.8 days. Peripheral platelet count increments in patients following platelet transfusions with concentrates stored 4 to 7 days in the new plastic were comparable to increments following transfusion of platelets stored 2 to 3 days in the other plastics studied. Bleeding times shortened in three of four patients receiving platelet concentrates stored from 4 to 6 days in the new plastic. Platelet concentrates stored in the new bag at 20 to 24 degrees C with flat-bed or elliptical agitation could be transfused for up to 5 days following phlebotomy with acceptable clinical results. The new plastic container is promising for storage of platelet concentrates for up to 7 days. Due to the higher pH of 50-ml platelet concentrates stored in bags made with the new plastic, the concentrates were superior at any storage interval to those stored in bags made of the other plastics studied.     

Plasma levels of ionized and total calcium during storage of citrated platelet concentrate

Measurements of ionized and total calcium levels in supernatant plasma samples from citrated platelet concentrates (PCs) were made over 7 days of storage. Both ionized and total calcium increased significantly during the storage period: respectively, from 0.074 mM Ca2+ in fresh platelet-rich plasma to 0.084 mM in PCs stored for 7 days (p = 0.017), and from 1.94 mM total calcium to 2.06 mM (p = 0.014) over the same period. The increase in calcium was partially blocked by the addition of platelet activation inhibitors to the PCs. Platelet-poor plasma stored under similar conditions showed no significant change in ionized or total calcium, which indicated that the increases observed in PCs were due to the release of cellular calcium. Significant correlations (p less than 0.01) were found between ionized or total calcium levels and lactate concentration or pH, but not hypotonic shock recovery rate. The demonstration of non-zero levels of ionized calcium makes it likely that Ca2+-dependent enzyme systems such as calpain expression and thrombin generation are active in the plasma of citrated PCs and may contribute to the platelet storage lesion.     

Plasma free fatty acid metabolism during storage of platelet concentrates for transfusion

New containers allow storage of platelet concentrates (PC) at 22 degrees C for up to 7 days, during which glycolytic and oxidative metabolism is vigorous. Recent evidence suggests that 85 percent of adenosine triphosphate regeneration is based on oxidative metabolism and that substrates other than glucose may be used. Because platelets can oxidize free fatty acids (FFA) as a possible source of energy during storage, the authors studied their availability, distribution, and turnover. Plasma FFA concentration was unchanged after 1 day of PC storage but significantly increased on Days 3, 5, and 7. Platelet-free plasma (PFP) stored under the same conditions as PC demonstrated a progressive increase in FFA, suggesting that some of the FFA accumulating in PC were derived from plasma rather than platelets. Indeed, during PC storage, plasma triglycerides decreased significantly, suggesting that they are a possible source of the increased levels of FFA found on Day 3 and thereafter. Thus, PC have a plasma FFA pool available continuously for oxidation during storage. Studies with radiolabeled palmitate suggested that FFA oxidation by platelets occurs during storage. The current findings show that plasma FFA could be a significant substrate for oxidative metabolism during storage of PC and that the oxidized FFA are replenished at least in part from plasma. These results may allow platelet storage to be improved, particularly in synthetic media.     

The effect of the interruption of agitation on platelet quality during storage for transfusion

BACKGROUND: A considerable amount of data and the CFR suggest that platelet concentrates (PCs) should be stored with continuous, gentle agitation before transfusion. However, there are only limited data concerning the mechanisms of platelet damage that may occur when agitation is interrupted, and there are no CFR guidelines concerning shipment between periods of storage. STUDY DESIGN AND METHODS: PCs were prepared by the platelet-rich plasma method and stored for 5 days at 20 to 24 degrees C; agitation was interrupted for 1 to 3 days either by simply stopping the agitator or by placing the PCs in a stationary shipping container. Measurements of platelet metabolism and quality were made during storage and on Day 5. RESULTS: With interruption on the agitator, the production of lactic acid was increased during the interruption in proportion to the number of platelets in the PC and the duration of the interruption. The pO(2) was increased during agitation interruption, which suggested a decline in oxygen utilization. With the use of the hypotonic shock response and the extent of shape change as reflections of platelet quality, there was no evidence of platelet damage unless the pH fell to or below 6.5. No PC reached this level after an interruption of agitation for only 1 day, irrespective of which day was chosen for interruption. PCs whose agitation was interrupted for 2 and 3 days were at risk of having a pH less than 6.5 if their contents were greater than 1.25 x 10(11) and 0.75 x 10(11) platelets, respectively. Interruption of agitation for 1 day in the shipping container produced results essentially identical to those produced by interruption on the agitator. CONCLUSION: Interruption of agitation of PCs for 1 day, either on the agitator or in the shipping container, produces no platelet damage measurable by these in vitro techniques. However, an interruption of agitation for 2 days can result in significant damage in some components. Further studies will be required to learn more about the mechanisms that lead to the metabolic changes described and to determine if the same generalizations apply to apheresis PCs and PCs prepared from pooled buffy coats.     

Loss of high-affinity thrombin receptors during platelet concentrate storage impairs the reactivity of platelets to thrombin

BACKGROUND: The storage of platelet concentrates (PCs) induces a reduction in the platelet surface expression of glycoprotein (GP) Ib alpha. The location of the platelets' high-affinity binding site for thrombin has been postulated as being located on GPIb alpha. This study attempts to determine whether loss or alteration of GPIb alpha during storage of PCs is related to impairment in the reactivity of platelets to thrombin. STUDY DESIGN AND METHODS: In this study, platelet surface expression of GPIb alpha was monitored by means of flow cytometry, throughout standard storage of PCs for up to 10 days. Two thrombin- induced platelet responses, the binding of radiolabeled fibrinogen and the platelet surface expression of P-selectin, were evaluated. Thrombin- binding assays were also performed to assess the number of thrombin receptors in platelets. RESULTS: The surface expression of the GPIb/IX complex declines during storage of PCs. The thrombin-induced maximal binding of fibrinogen in platelets stored for 3, 7, and 10 days was 77 +/? 7 percent, 60 +/? 20 percent, and 34 +/? 25 percent, respectively, of that found in fresh platelets. Moreover, the concentration of thrombin needed for 50 percent of platelets to express the CD62 antigen P-selectin at the surface increased from 0.05 U per mL in fresh platelets to 0.11, 0.56, and 1.2 U per mL in platelets stored for 3, 7, and 10 days, respectively. Thrombin-binding experiments demonstrated a significant reduction in the number of high-affinity binding sites throughout storage of PCs (55 +/? 21 sites/platelet in 10-day-stored platelets vs. 73 +/? 25 in fresh platelets). A significant correlation was also observed between the number of high-affinity thrombin-binding sites and surface expression of GPIb alpha. Selective blockage of the thrombin-binding site on GPIb alpha with monoclonal antibody LJ-Ib10 also inhibited the response of fresh platelets to thrombin, up to a level equivalent to that found in 3-day-stored platelets. CONCLUSION: The loss of the GPIb alpha-located high-affinity thrombin-binding site may impair the ability of platelets to become activated by thrombin as storage time increases.     

Quantitation of phosphatidylserine-exposing platelets in platelet concentrate prepared in routine blood transfusion laboratory

BackgroundPhosphatidylserine (PS) plays important roles in platelets’ pro-coagulant function. However, little is known about assessing this molecule in platelet concentrates (PCs) prepared for routine blood transfusion service.AimTo quantitate the number of PS-exposing platelets in PCs prepared in a routine transfusion laboratory.MethodsPC products were prepared according to routine laboratory procedure. The numbers of PS-exposing platelets in the PCs and in unprocessed whole blood were determined using flow cytometry.ResultsA cross-sectional study of 253 PCs found that they had significantly increased numbers of PS-exposing platelets compared to unprocessed whole blood (47,439 ± 26,500 cells/μL; 5903‒166,156 cells/μL) vs. 30,058 ± 12,958 cells/μL; 8,154-86,606 cells/μL). A heterogeneity study demonstrated that 6% and 2% of the measured PCs and of unprocessed donor whole blood, respectively, showed an increase in the number of PS-exposing platelets that was greater than 2 fold.ConclusionsThe study suggested that the number PS-exposing platelets in PC prepared in a routine transfusion laboratory differs. However, assessment of the number of PS-exposing platelets in platelet products could be a valid measure to use in managing the quality of platelet processing in routine laboratories.     

Extension of platelet concentrate storage by addition of sodium bicarbonate

Viability of platelet concentrate (PC) stored in polyvinylchloride bags in an elliptical rotator at 22 degrees C (standard PC) was assessed by in vitro tests, and an alternate approach to extending the shelf-life of PC by the addition of hypertonic sodium bicarbonate (test PC) was investigated. The fall in the pH which occurred during storage in standard PC was arrested in test PC. Furthermore, platelets stored under these test conditions maintained their morphology better than in standard PC as judged by their mean platelet volume and platelet distribution width. Recovery of stored platelets from hypotonic shock at 37 degrees C following resuspension in fresh plasma was better for test platelets. Results indicated that platelets in standard PC were viable up to day 3 but were not viable at day 7. Platelets store better in PC to which sodium bicarbonate has been added and behave as viable platelets up to 7 days.     

Fatal Salmonella septicemia after platelet transfusion

A thrombocytopenic, leukopenic patient with multiple myeloma who was given 7 units of platelets died 6 days later from complications of Salmonella heidelberg septicemia. A platelet donor who was asymptomatic at the time of donation had group B Salmonella on stool culture. His clinical history and the results of serologic studies and stool culture were consistent with a mild Salmonella gastroenteritis 5 days before donation. Antibiotic sensitivity patterns and plasmid profiles indicated that the organism (S. heidelberg) isolated from the donor's stool was identical to that isolated from the patient's blood and from the platelet bags. It is believed that low-grade, asymptomatic bacteremia in the donor was the source of infection in the recipient. Food and Drug Administration records contain reports of six septic deaths due to platelet transfusions since 1979, compared with none in the preceding 4 years. Increased use of platelet products and the standard practice of storage at room temperature may contribute to the risk of sepsis after platelet transfusion, particularly in immunocompromised patients.     

Quantitation of coated platelet potential during collection, storage, and transfusion of apheresis platelets

BACKGROUND: Coated platelets (PLTs), a subpopulation of PLTs observed upon dual agonist stimulation with collagen and thrombin, are known to retain several procoagulant α‐granule proteins on their surface. By formation of a highly active membrane‐bound prothrombinase complex, these PLTs represent an important step in the coagulation cascade as a consequence of their ability to generate thrombin at the site of vascular injury. Various clinical observations suggest that higher levels of coated PLTs are associated with thrombosis while a deficiency of coated PLTs results in a bleeding diathesis. Current quality control guidelines for in vitro PLT storage measure PLT viability but no routine evaluation of the hemostatic function of stored PLTs and particularly no estimation of coated PLT potential is performed. Our primary objective was to evaluate if the process of apheresis and storage of PLT units alters the levels of coated PLTs. In addition, we sought to determine how transfusion of stored PLTs into patients with thrombocytopenia affects the patient's coated PLT levels. STUDY DESIGN AND METHODS: Coated PLT levels were analyzed in 13 voluntary PLT donors before donation, in the fresh apheresis product (Trima, CaridianBCT) and in the stored apheresis product just before transfusion. In addition, 10 patients with thrombocytopenia were analyzed for coated PLTs before and after transfusion of a stored PLT product. RESULTS: Coated PLT levels were significantly decreased after the process of apheresis (17% relative decline; p < 0.01) and with prolonged storage (1 to 5 days; 53% relative decline; p < 0.001). Transfusion of stored PLT units did not result in significant increment of coated PLT levels in patients with thrombocytopenia as expected considering the low level of coated PLTs in stored PLT units. Furthermore, there was no suggestion of regeneration of coated PLT potential upon reinfusion. CONCLUSIONS: Isolation and storage of apheresis PLTs by standard blood bank procedures results in a significant decline in coated PLT potential. Reinfusion of stored apheresis PLTs into patients with thrombocytopenia resulted in a predictable change in coated PLT potential with no suggestion of regeneration of lost coated PLT potential.     

Extension of platelet concentrate storage to 7 days in second- generation bags

Platelet concentrates stored for 7 days in 50 ml of plasma in both thin film and enlarged variations of the standard 5-day CLX plastic bags were evaluated for pH maintenance and in vivo viability by two laboratories working independently. 51Cr-labeled platelets were reinfused into normal volunteers at the end of storage and recovery and half-life calculated. The pH was maintained well; less than 10 percent of units fell below 6.0 at 7 days. Mean 7-day recovery for both laboratories was 43.6 +/- 11.6 percent in the thin-film bag and 45.4 +/- 8.52 percent in the enlarged bag, compared with 43.6 +/- 8.8 percent at 5 days in the 5-day plastic licensed bag. After 7 days storage the half-life was 3.6 +/- 0.9 days in the thin-film bag and 3.7 +/- 0.6 days in the enlarged bag, compared with 3.6 +/- 0.5 days in the previously licensed CLX plastic bag after 5 days. Thus, platelet viability was maintained well at 7 days of storage in both of the container variations that allowed increased gas exchange.     

Progressive platelet activation with storage: evidence for shortened survival of activated platelets after transfusion

Platelets are known to become activated during storage, but it is unclear whether such activation affects recovery or survival after platelet concentrate (PC) transfusion. With the use of flow cytometry to determine the percentage of platelets expressing the alpha-granule membrane protein 140 (GMP-140), a known adhesive ligand appearing on the platelet surface after activation, several studies were conducted. These investigations evaluated 1) the occurrence of significant platelet activation over time in PCs (n = 46) stored under standard blood bank conditions; 2) the correlation between platelet activation and platelet recovery in normal subjects after PC storage (n = 12), as assessed by the recovery of Indium-labeled platelets; and 3) the recovery of activated and unactivated platelets in thrombocytopenic cancer patients transfused with standard PCs (n = 11). It was determined 1) that an increasing duration of storage of PC was associated with increasing platelet activation as measured by the percentage of platelets expressing GMP-140, progressing from a mean of 4 +/- 2 percent (SD) on the day of collection to a mean of 25 +/- 8 percent by 5 days of storage: 2) that, in normal subjects, posttransfusion recovery of autologous platelets stored for 2 to 4 days and then labeled with In111 was inversely correlated with the percentage of activated platelets in the transfused PC (r = -0.55, p = 0.05); and 3) that, when thrombocytopenic patients were transfused with standard PCs, the recovery of the activated platelets in the transfused PCs averaged only 38 +/- 15 percent of the number predicted by the absolute platelet increment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic changes during platelet storage

Platelet concentrates (PC) were stored in plastic bags with continuous shaking at 4, 22, and 37 C. Various metabolic parameters were examined over a 72-hour period. At 22 C, the pH and PO2 declined over 72 hours while the PCO2 and lactate increased. Hypotonic shock declined to 70 per cent. This differed from the small amounts of CO2 and lactate found at 4 C and the marked accumulation of metabolites and almost complete loss of shock response at 37 C. Aggregation was always better maintained with 4 C storage. The toxic effect of the accumulation of metabolites on the platelets was tested by adding lactate to fresh PC at zero time. This was effective in lowering the initial pH, markedly inhibiting the response to aggregation and decreasing the total accumulation of lactate during storage, but did not produce an inhibition of hypotonic shock response. The effect of accumulation of toxic metabolites was further investigated by using 72-hour plasma and platelets and recombining each of them with fresh preparations. Platelets were tested under degassed conditions to outline the requirements for oxygen and gasious exchange. Surprisingly, there was less accumulation of lactate and CO2 and better hypotonic shock response. These experiments have detected various changes in viability markers in platelets that are stored under actual blood bank conditions and indicate that the accumulation of lactate is not totally responsible for the toxic inhibition of platelet performance that is found upon storage at 22 C.     

Quality control of platelets during storage by the PFA-100: a comparison to platelet aggregation.

BACKGROUND AND OBJECTIVES: The PFA-100 examines platelet function by measuring closure time (CT) when whole blood is passing through a capillary and a filter membrane coated with a collagen/platelet agonist. Termination of the CT is achieved when adhered and aggregated platelets of the passing blood flow occlude the membrane. Two PFA-100 test cartridges are used to measure the CT of citrated whole blood; either the collagen/ADP (PCA) or the collagen/ epinephrine (PCE). We investigated the applicability of the PFA-100 test system for quality control of platelet concentrates in comparison with platelet aggregometry. MATERIALS AND METHODS: For platelet aggregation, the combination of ADP (100 microM/l) and collagen (20 microg/ml) were used as agonists (ADP/COL). In our test system, 25 microl epinephrine (10(-3) M) were added to the PCA cartridge (CT: PCAE) and 25 microl ADP (100 microM/l) to the PCE cartridge (CT: PCEA), respectively. Red blood cells from a blood group 0 donor were adjusted to a hematocrit of 43% using platelet rich plasma (8 x 10(5)/microl) of the respective platelet concentrate. Fourteen irradiated and non-irradiated platelet concentrates were examined on days 0, 3 and 5 after platelet preparation. Swirling without a scoring system and mean platelet volume (MPV) were also tested. Statistical analyses were performed by Pearson's range coefficient, the Mann and Whitney test, and the Wilcoxon test. RESULTS: Swirling was seen in all platelet concentrates. Mean platelet volume was normal during the whole observation period like the PCAE in non-irradiated products. Only the PCAE showed finite CTs during the whole storage time. Consequently, further testing was performed exclusively with the PCAE. Weak but significant correlations could be found between the PCAE and ADP/COL (r = -0.53 for non-irradiated platelet products, r = -0.62 for irradiated platelet products, p < or = 0.0001). Platelet function significantly decreased to around 60% of the initial value in the PCAE on day 5 of storage, ADP/COL decreased to 35% of maximum amplitude. A further decrease of approximately 10% was observed in the irradiated products, a fact which was significant in the PCAE. Only in the irradiated platelet products did MPV show a weak but significant correlation with the PCAE (r = 0.45, p = 0.002). CONCLUSION: Functional features of platelets from platelet concentrates characterized by ADP/COL induced aggregation can also be measured by the PFA-100. Yet, it has a higher sensitivity to platelet lesions induced by gamma-radiation. Further clinical studies will be necessary to determine whether PFA-measurements meet the quality criteria of MPV or swirling, or is superior in predicting in vivo viability. The relatively high cost of the test cartridge remains an obstacle for routine use.