The effect of filtration on residual levels of coagulation factors in plasma.

Leukoreduced blood components are commonly manufactured via filtration. There are specifications for the residual leukocyte content of any final cellular blood component but not for residual clotting factors. Leukoreduced and nonleukoreduced platelet-poor plasma products were manufactured from filtered vs unfiltered platelet-rich plasma, respectively, using platelet leukoreduction filters. The leukoreduced plasma showed lower levels of factor VIII (75% ± 16% vs 88% ± 18%, P ≤ .05), factor XI (86% ± 9% vs 96% ± 10%, P ≤ .01) and factor VII (87% ± 14% vs 98% ± 11%, P ≤ .01). No difference was seen with factor X, factor V, or fibrinogen. Plasma filtered through a whole blood filter showed a reduction in factor V (105% ± 12% vs 124% ± 10%, P ≤ .01) but a minimal reduction in factor VIII (80% ± 5% vs 82% ± 6%, P = .04). Filtration can alter the residual levels of clotting factors to a variable extent in manufactured plasma, most noticeably factors V, VII, VIII, and XI.

Manufacture of pooled platelets in additive solution and storage in an ELX container after an overnight warm temperature hold of platelet-rich plasma.

The processing of whole blood-derived platelet-rich plasma (PRP) to a platelet concentrate and platelet-poor plasma is currently performed within 8 hours to comply with the requirements to manufacture fresh frozen plasma. Maintaining PRP at room temperature for a longer period can have the advantage of shifting the completion of component manufacture onto day shifts. Pairs of ABO-identical prepooled platelets were manufactured by the PRP method, using the current approach with platelet storage in a CLX HP container (Pall Medical, Covina, CA) and plasma, or a novel approach with an 18- to a 24-hour room temperature hold of the PRP and the manufacture of pooled platelets in a glucose-containing additive solution (AS) and storage in a new ELX container (Pall Medical). Standard in vitro assays were performed on days 2, 5, and 7. The results showed that the AS platelets in ELX have in vitro characteristics that are equivalent or superior to those of the standard product.

Evaluation of the properties of components prepared and stored after holding of whole blood units for 8 and 24 hours at ambient temperature.

BACKGROUND: The capability of holding whole blood (WB) units at ambient temperature, overnight, should help in platelet (PLT) concentrate preparation logistics. We summarize the results of a study conducted in the early 1990s that compared, in particular, PLT and red blood cell (RBC) in vivo viability properties following storage after preparation after 8- and 24-hour WB hold periods. STUDY DESIGN AND METHODS: Individuals donated units of WB on two occasions. Centrifugation at 20 to 24°C to separate PLTs and additive system RBC placement at 1 to 6°C was completed 8 hours after phlebotomy or after 24 hours in randomized order. Components were not leukoreduced. Studies including in vitro biochemical and hematologic analyses and autologous in vivo RBC and PLT evaluations were conducted at two sites. RESULTS: RBC 24-hour in vivo (mean ± SD) recoveries (single-label approach), after 35 days of storage, were 79.2 ± 4.3 and 79.4 ± 3.9% (n = 9; p > 0.05), with WB holding periods of 8 and 24 hours, respectively. With 42 days of storage, recovery after a 24-hour hold was slightly less than with an 8-hour hold (72.9 ± 6.5% vs. 76.0 ± 5.4%; n = 17; p < 0.05). RBC 2,3-diphosphoglycerate acid levels were substantially less after the 24-hour hold compared to after the 8-hour hold (n = 18; p < 0.05). PLT recovery after 5 days of storage with 8- and 24-hour hold periods were similar, 51.1 ± 14.9 and 50.6 ± 17.7%, respectively (n = 18; p > 0.05). The PLT survival variable and in vitro properties reflecting storage quality also showed no significant difference. CONCLUSION: RBC and PLT in vivo variables, and most in vitro variables, were not significantly different after storage with WB holding times of 8 and 24 hours except for a slight diminution of RBC recovery with the 24-hour hold after 42 days of storage.

Stability of coagulation factors in plasma prepared after a 24-hour room temperature hold.

BACKGROUND: The manufacture of fresh-frozen plasma (FFP) requires that plasma be frozen within 8 hours of collection and 24-hour frozen plasma requires 1 to 6°C refrigeration before freezing. Manufacture of plasma after a room temperature hold for 24 hours, while convenient, could compromise clotting factor levels. STUDY DESIGN AND METHODS: Pairs of FFP and 24-hour room temperature-frozen plasma (PLT-rich plasma [PRP]-24HRTFP) were manufactured from PRP after a room temperature hold for 8 and 24 hours, respectively. Additional whole blood (WB) donations were kept at room temperature for 24 hours before plasma manufacture (WB-24HRTFP). The frozen plasma products were stored at -18°C, thawed, and then stored at 1 to 6°C, with coagulation factor assays performed for up to 7 days. RESULTS: On the day of thaw, Factor (F)VIII was lower in PRP-24HRTFP by 13% (p = 0.002) but not in WB-24HRTFP (p = 0.3) compared to FFP. All other clotting factors were within normal range. During the postthaw period FVIII and FV declined 25 and 6%, respectively, in WB-24HRTFP and 23 to 50% in the paired products; however, the difference between both types of 24HRTFP and FFP is insignificant by Day 7 (p > 0.05). Other clotting factors either were unchanged or showed minimal reduction (< 15%). CONCLUSION: Plasma manufactured after a 24-hour room temperature hold contains coagulation factors comparable to FFP except for a possible reduction of up to 20% in FVIII. This plasma appears suitable as a transfusable product and extension of liquid storage to 7 days merits consideration.

In vitro assays used in the evaluation of the quality of stored platelets: correlation with in vivo assays.

There have been reports on platelet storage studies that have demonstrated little or no correlation of in vitro parameters with in vivo parameters of viability such as CCI, and % recovery and survival by radio labeling. However, this does not mean that the in vitro parameters necessarily are poor predictor of in vivo viability, but may be related to the fact that in the vivo parameters, as absolute values, do not precisely reflect the viability of the platelets due to uncertainty in the splenic uptake and inaccuracy of the estimated blood volume of the recipient. A more informative way to examine the predictability of the in vitro parameters is to examine whether processing and storage conditions associated with loss or improvement in vivo viability are also associated with corresponding changes to the in vitro parameters and vice versa.

Detection of bacteria in stored red cell products using a culture-based bacterial detection system.

BACKGROUND: The Pall eBDS uses oxygen consumption as a surrogate marker for bacterial detection in platelet (PLT) products. This article describes the evaluation of eBDS to detect bacterial contamination in inoculated fresh and stored leukoreduced red cell (RBC) units. STUDY DESIGN AND METHODS: Field studies were conducted at three sites to establish eBDS pouch incubation time and the pass/fail threshold. RBC units were inoculated with each of 12 bacterial species known to cause sepsis at target inocula of either 1 to 15 or 100 colony-forming units (CFUs) per mL. Units were mixed and stored at 2 to 6 degrees C. Samples were taken for culture and eBDS testing weekly from 0 hour to 42 days and incubated for 35 degrees C for 24 to 30, 48, and 72 hours, followed by measurement of percent oxygen content. RESULTS: The studies showed growth of five bacterial species (including Yersinia enterocolitica) in RBC units, while seven bacterial species showed no growth or autosterilized. A pass/fail oxygen threshold of 14.4 percent was determined based on results from noninoculated controls (n = 633) and from inoculated samples (n = 884) after 48 hours of incubation. Detection was 100 percent at all sampling times during refrigerated storage with both 48 and 72 hours of pouch incubation. CONCLUSION: With incubation of eBDS pouches for 48 and 72 hours, 100 percent detection was obtained in 884 samples with bacterial levels of at least 1 CFU per mL, and no false-positive samples were obtained. Based on the bacterial growth patterns, RBC units may be sampled 1 to 3 days after collection for optimal efficacy and read after 48 to 72 hours of incubation of the eBDS sample pouches at 35 degrees C. The Pall eBDS is suited for detection of typical bacterial contaminants in fresh and stored RBCs.

Prediction of red cell and blood volumes distribution by various nomograms: do current nomograms overestimate?

BACKGROUND: Currently used formulas for estimation of a person's red cell volume (RCV) by weight and height are decades old and were based on the use of 51Cr isotopes and on a sample population, which may not be reflective of today's population. In this study, the accuracy and precision of the use of 99mTc RCV measurements in volunteers more typical of today's population were evaluated. STUDY DESIGN AND METHODS: The subjects were volunteers who met the requirements for a standard blood donation. RESULTS: The mean +/- standard deviation (SD) 99mTc RCV for 127 males (mean weight, 83.2 kg; height, 180 cm) was 2062 +/- 339 mL, and for 101 females (mean weight, 69.5 kg; height, 166 cm) it was 1320 +/- 201 mL. These results were highly correlated with RCV results with the standard extrapolation 51Cr method with stored red blood cells (RBCs) and highly consistent (within +/-10%) by repeated measurements with the same 22 donors over a 3.5-year period. The RCV results correlated with estimates from the current formulas, but were on average 11 to 14 percent lower. CONCLUSION: The studies demonstrated that 99mTc is a reproducible and precise method for determination of a person's RCV and that current formulas may significantly overestimate the RCV of today's population. This is likely the result of a shift in population characteristics over the past four decades as reflected by an increased mean body mass index (from 25 to 28 kg/m2), which has not resulted in a proportionally increased RCV.

In vitro evaluation of prestorage pools consisting of mixed A and O platelet concentrates.

BACKGROUND: Current FDA regulations allow the prestorage pooling of whole blood-derived platelet concentrates (PCs) of identical ABO type in a recently cleared platelet (PLT) pooling bag (Acrodose, Pall Medical). It is unclear how ABO-mixed PC pools would store if pooled before storage. STUDY DESIGN AND METHODS: Pools consisting of ABO-identical PLTs and mixed A and O PLTs in varying proportions were evaluated on Days 1, 5, and 7 of storage with measures of the PLT storage lesion, lymphocyte activation, and activation of the complement and coagulation system. Data were analyzed by analysis of variance. RESULTS: Pools did not differ on Day 7 in pH (p = 0.63), hypotonic shock response (p = 0.25), extent of shape change (p = 0.26), morphology score (p = 0.18), or white cell count (p = 0.79), but surface P-selectin expression was more evident in the ABO-mixed pools (p = 0.02). Small microscopic clumps of PLTs were observed in all pools, but were more prominent in the ABO-mixed pools (p < 0.01). PLT counts, however, did not differ between pools (p = 0.93), indicating that only a small proportion of PLTs were clumped. Surface A-antigen expression was proportional to the number of A PCs in each pool and did not vary between study days. Anti-A(1) titers were either unchanged or decreased by one dilution. Complement and coagulation activation markers did not differ between pools. CONCLUSION: Pooling A and O PCs is associated with evidence of increased microscopic PLT clumping and activation, but these differences are not exacerbated with 7-day storage. Other major measures of PLT quality do not differ, and there was no evidence of a mixed lymphocyte reaction.

In vitro pH effects on in vivo recovery and survival of platelets: an analysis by the BEST Collaborative.

BACKGROUND: The pH environment of stored platelet (PLT) products is recognized as an important factor and is generally used as a key surrogate measure of PLT viability. It is the only in vitro measurement that has been translated into industry standards and regulatory rules or specifications for storage of PLT products. The objective of this study was to evaluate the effect of in vitro pH on the in vivo recovery and survival of autologous PLT products. STUDY DESIGN AND METHODS: Data from individual autologous radiolabeled PLT kinetic studies were solicited from independent laboratories. PLTs stored for at least 5 days in 100 percent autologous plasma with a pH(22 degrees C) of at least 6.2 were analyzed. Data were fit to a mixed-effects regression model with fixed effects of pH(22 degrees C), time of storage, and preparation method-storage bag combination. RESULTS: Eight research laboratories reported 476 individual recovery and survival results with associated pH before labeling from a variety of autologous, radiolabeled PLT kinetic studies from September 1999 to March 2005. These results are from 254 individual subjects who donated a total of 386 PLT units, with up to nine collections per subject reported. The effect of pH on either PLT recovery (p = 0.86) or survival (p = 0.55) was not significant. Time of storage and the method-bag combination both had significant effects on these outcomes (p < 0.0001). CONCLUSION: These data suggest that there is no relationship between in vitro pH at a pH(22 degrees C) of at least 6.2 and in vivo PLT viability as measured by radiolabeled recovery and survival of autologous PLTs.

Storage of platelet-rich plasma-derived platelet concentrate pools in plasma and additive solution.

BACKGROUND: Prestorage pooling of platelet (PLT)-rich plasma (PRP)-derived PLT concentrates (PCs) and storage in either plasma (PS) or an additive solution (AS) is logistically feasible and would result in a product similar to buffy-coat or apheresis PLTs. STUDY DESIGN AND METHODS: On Day 0, PS PRP PCs were pooled with a sterile connecting device into a new 1.3-L storage container (ELX, PALL Medical). AS-PCs were prepared by addition of a new low-pH glucose-containing AS to the PLT sediment. AS-PCs were pooled into a 1.3-L ELX bag containing four tablets of NaHCO3. PC pools were sampled on Days 1, 5, and 7. RESULTS: PS pools containing 5 units had a mean PLT yield similar to the AS pools (39 x 10(10) +/- 3 x 10(10) vs. 37 x 10(10) +/- 6 x 10(10); p = 0.25). All pools had WBC counts of less than 1 x 10(6). pH and HCO3 decreased in PS pools with storage, but either increased or remained constant in the AS pools. On Day 7, no differences were seen in morphology score or extent of shape change. Hypotonic shock response was better preserved in the plasma pools (71 +/- 12% vs. 56 +/- 13%, p < 0.01); however, surface P-selectin was expressed less in the AS pools (6 +/- 4% vs. 18 +/- 10%, p < 0.01). CONCLUSION: Manufacture and storage of PRP-PCs in pools either in plasma or in a glucose-containing AS in this new container are feasible, and there is good preservation of PLT quality to Day 7.

In vitro and in vivo evaluation of leukoreduced platelets stored for 7 days in CLX containers.

BACKGROUND: Extension of platelet (PLT) storage and concomitant use of a bacterial detection system would provide logistical advantages by reducing outdating and improving patient care through promotion of the use of sensitive detection systems. This study evaluated the in vitro characteristics and in vivo viability of leukoreduced PLT units derived from PLT-rich plasma stored for 5 days (control) versus 7 days (test) in CLX plastic containers. STUDY DESIGN AND METHODS: Two whole-blood units were collected from each subject into a leuko-reduction filtration system (Leukotrap RC-PL system, Pall Medical) in a paired design, the second 2 days after the first. These were leukoreduced (Leukotrap PL) and stored for 7 and 5 days. Poststorage samples from test and control units were randomly labeled with (51)Cr or (111)In and simultaneously infused autologously to determine recovery and survival. RESULTS: Small but significant (p < 0.05, paired t test) differences between 5 and 7 days of storage were seen in in vitro variables such as extent of shape change, hypotonic shock response, morphology, and P-selectin expression. In vivo recovery declined on average 11 percent with the two additional days of storage from 54.4 +/- 13.6 to 48.7 +/- 15.0 percent (p < 0.002); survival decreased on average 19 percent from 6.7 +/- 1.0 to 5.4 +/- 1.7 days (p < 0.002). CONCLUSION: Storage for 7 days was associated with reduced recovery and survival and in vitro variables, suggestive of extension of the storage lesion. These differences, however, were small in magnitude and unlikely to have significant clinical effects. Current collection and storage systems provide PLTs that are as functional at 7 days as those licensed for 7-day storage two decades ago.

Current issues related to the quality of stored RBCs.

This paper reviews some current issues related to the quality of red cells processed and stored for transfusion. These include the storage lesion, use of gamma irradiation, and storage of red cells in containers of plastic made with non-leachable plasticizers.

Enhancement of a culture-based bacterial detection system (eBDS) for platelet products based on measurement of oxygen consumption.

BACKGROUND: An enhanced bacterial detection system (Pall eBDS) was developed that distinguishes itself from its predecessor (Pall BDS) by removal of the platelet (PLT)-retaining filter allowing for optimal bacterial transfer, modification of the culture tablet to reduce the confounding effects of respiring PLTs while enhancing bacterial growth, and facilitation of nutrients and gas exchange by agitating the sample pouch during incubation at 35 degrees C. The objective was to evaluate the performance of the new eBDS. STUDY DESIGN AND METHODS: Leukoreduced whole blood-derived PLT concentrates (LR-PCs) and LR single-donor PLTs (LR-SDPs) were inoculated with 1 to 15 colony-forming units (CFUs) of bacteria per mL in studies of each of 10 bacterial species associated with fatal transfusion-transmitted bacterial infection. Immediately after inoculation and after 24 hours of storage at 22 degrees C, samples of inoculated LR-PCs were aseptically transferred into the eBDS pouches. Pouches were then incubated for 24 hours at 35 degrees C with agitation and oxygen concentration was then measured. RESULTS: Median inoculation levels ranged from 5 to 13 CFUs per mL for each species studied. No significant differences in oxygen concentration were found when comparing LR-PCs with LR-SDPs. When sampling occurred from the PLTs 24 hours after inoculation, all 280 cases (24-33 replicates of each species) were detected as contaminated by the device (100% sensitivity). No false-positives were obtained with 713 uninoculated PLT units. CONCLUSIONS: The eBDS demonstrated improved detection sensitivity in the range of 1 to 15 CFUs per mL with no observed false-positives compared to the original BDS (detection range 100 to 500 CFUs/mL) with no false-positives.

Prestorage pooled whole-blood-derived leukoreduced platelets stored for seven days, preserve acceptable quality and do not show evidence of a mixed lymphocyte reaction.

BACKGROUND: Prestorage pooling of whole-blood-derived PCs (WBD-PCs) would be advantageous to transfusion services in that it would make the product available in a more timely manner, reduce wastage of untransfused pools, and simplify bacterial screening by allowing testing of the pool rather than each single PLT concentrate (PC). STUDY DESIGN AND METHODS: Four to six individual leukoreduced PCs were pooled into a 1.5-L CLX-HP PLT storage bag using a sterile connecting device. Controls were individual prestorage leukoreduced PCs that were stored as single products. Products were sampled on Days 5 and 7 for measures of PLT quality; coagulation, fibrinolytic and complement activation; and for evidence of a mixed lymphocyte reaction. RESULTS: The pH level was well maintained to Day 7 with no prestorage pool having a pH below 6.7. Day 7 studies showed no evidence of coagulation or difference in complement activation. F1.2 levels did not differ between Days 5 and 7, but a 10- to 15-percent increase in C3a des-Arg was observed between these days in all product types. Day 7 activated lymphocyte surface markers (CD69, CD71, HLA-DR) were all at lower limits of detection in the prestorage pooled products, and levels of supernatant cytokines were either not different between product types on either study day or, if different, were lower in the prestorage pooled products. CONCLUSION: There is no evidence of a deterioration in quality, activation of coagulation or complement, or a mixed lymphocyte reaction attributable to the prestorage pooling process with up to 7 days of storage.

Detection of bacteria in WBC-reduced PLT concentrates using percent oxygen as a marker for bacteria growth.

BACKGROUND: The risk of receiving a PLT concentrate (PC) contaminated with bacteria may be 1000-fold greater than that of pathogenic viral transmission, yet surveillance for this risk is not generally practiced. A novel bacteria detection system (BDS) that overcomes the limitations of current systems is described. The BDS monitors percent oxygen (%O2) in air above aliquots of PCs that have been filtered to remove the confounding effect of respiring PLTs and residual WBCs. STUDY DESIGN AND METHODS: One-day-old WBC-reduced whole-blood-derived PCs (WBPCs) were inoculated with bacteria at 100 to 500 CFU per mL. After 30 minutes, 2- to 3-mL aliquots were processed through a PLT-reducing filter into a sample pouch containing sodium polyanethol sulfonate and entrained air. After incubation at 35 degrees C for at least 24 hours, the %O2 was measured within the pouch. Noninoculated WBC-reduced WBPCs (n = 155), confirmed free of bacteria by routine culture, were tested in a like manner. Results from the latter group of WBC-reduced WBPCs were used to distinguish contaminated from noncontaminated units. RESULTS: After a 24-hour incubation at 35 degrees C, 195 (96.5%) of the 202 sample pouches obtained from inoculated units were detected by the BDS. After an additional 6 hours at room temperature, those that remained and were tested were found positive. None of the noninoculated controls produced a positive reading. CONCLUSION: The BDS is easy to use and provides good levels of sensitivity and specificity.