Postthaw stability of fibrinogen in cryoprecipitate stored between 1 and 6 degrees C

The fibrinogen activity in thawed cryoprecipitate stored between 1 and 6 degrees C is maintained essentially unchanged in most bags for a month. Occasionally, a bag will have a reduction in fibrinogen. If pooling has not occurred, thawed cryoprecipitate should be useful as a source of fibrinogen for a period of time considerably in excess of the 6 hours allowed for its use as a source of factor VIII or von Willebrand factor.     

Fibrinogen in cryoprecipitate and its relationship to factor VIII (AHF) levels

Very little has been published to indicate the quantity of fibrinogen in cryoprecipitates. We assayed 88 preparations from five blood banks for factor VIII(AHF) and fibrinogen to assess whether the AHF assay can predict the fibrinogen content. Cryoprecipitate was considered to be consistent with FDA standards with 80 units of factor VIII/bag (40% yield from 200 ml plasma). Fibrinogen was considered adequate if 200 mg were recovered (40% yield, 200 ml plasma, normal range 150–350 mg/dl). The mean AHF was 145 units/bag and fibrinogen. In 64/88 bags, the fibrinogen and AHF were concordant, but in 24/88 bags the results were discordant. Although it appears safe to conclude that a bag of cryoprecipitate will average 250 mg fibrinogen, adequate control may require separate assays for fibrinogen.     

Determinants of factor VIII recovery in cryoprecipitate

Many aspects of the production of cryoprecipitate were studied to determine which methods resulted in the greatest recovery of Factor VIII. The following recommendations resulted: 1) blood should be mixed with anticoagulant throughout phlebotomy; 2) blood should be centrifuged within a few hours of collection; 3) larger satellite bags should be used to contain the usual volume of plasma, for example, 200 ml of plasma should be frozen in a 600-ml capacity bag; 4) plasma should be centrifuged as soon as thawing is complete; 5) cryoprecipitate should be refrozen on dry ice; 6) cryoprecipitate should be stored at or below -30 C.; and 7) prolonged storage of frozen plasma or cryoprecipitate should be avoided. Variations in Factor VIII content from one bag of cryoprecipitate to another, under uniform production conditions, depends largely on two donor-specific attributes which tend to remain constant from time to time, namely, the donor's plasma Factor VIII level and the cryoprecipitability of his Factor VIII.     

Coagulation factor levels in plasma frozen within 24 hours of phlebotomy over 5 days of storage at 1 to 6 degrees C

BACKGROUND: The use of plasma frozen within 24 hours after phlebotomy (FP24) is likely to increase as male donors become the predominant source of plasma products. This study was performed to investigate the levels of clotting factors in thawed plasma (TP) prepared from FP24 during 5 days of storage at 1 to 6°C. STUDY DESIGN AND METHODS: Five units of A, B, and O and 3 units of AB FP24 were obtained from the local blood provider. They were thawed and maintained at 1 to 6°C for a total of 5 days. Within 6 hours of thawing and every 24 hours thereafter for 5 days, each unit was assayed for the following clotting factors: Factor (F)II, FV, FVII, FVIII, F IX, FXI, FXII, antithrombin (AT), protein C (PC), and protein S (PS). ADAMTS‐13 was assayed on Days 2, 4, and 5. Time is expressed as mean hours or days (standard deviation). RESULTS: On average the units were frozen 21.3 (3.8) hours after phlebotomy and had been frozen for a mean of 30.1 (32.3) days before thawing. The activities of all procoagulant factors including FVIII, along with AT, PC, and ADAMTS‐13, were well maintained in their normal range during the 5‐day storage. The activity of PS was slightly below the normal range by Day 5. CONCLUSIONS: The activity of all factors assayed, except for PS, were within their normal range during the 5‐day storage period. These results show comparable factor assay levels in TP prepared from fresh‐frozen plasma and FP24.     

Studies on platelets exposed to or stored at temperatures below 20 degrees C or above 24 degrees C

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.     

Electrophysiological and biochemical changes in rabbit hearts stored at 4 degrees C for 6 or 24 h

This study examines the electrophysiological and metabolic changes that occur in rabbit hearts during hypothermic storage in vitro. Hearts were microperfused at 4 degrees C for 6 or 24 h with either normal Krebs-Henseleit buffer (KHB) or KHB containing 2,3-butanedione monoxime (BDM). After hypothermic storage, hearts were rewarmed to 37 degrees C with KHB. Cardiac function was then assessed in Langendorff perfusion mode. Electrophysiological changes were also assessed from the ventricular paced-evoked responses. After storage, mitochondria were isolated from the hearts and their respiratory control ratio, rate of ATP synthesis and outer membrane intactness were assessed. Compared with values from fresh non-stored hearts, hearts stored hypothermically for 24 h showed significant decreases in both left ventricular developed pressure and coronary flow when reperfused in Langendorff mode. On the other hand, the decrease in left ventricular developed pressure in hearts that were stored for only 6 h (with or without BDM) was not significant. Compared with values obtained from fresh non-stored hearts, hypothermic storage significantly decreased the R-wave amplitude, and both the R-E and ST-E intervals of paced-evoked responses. This was true for hearts microperfused for 6 h (with or without BDM) and for hearts microperfused with buffer containing BDM for 24 h. The ST-R intervals in hearts microperfused hypothermically for 6 h were prolonged, but this change was not statistically significant compared with those obtained from unstored hearts. In hearts microperfused with KHB containing BDM for 24 h, the ST-R interval was significantly prolonged. Hypothermic microperfusion for 24 h significantly decreased both the mitochondrial coupling ratio and the rate of ATP synthesis. In hearts microperfused with BDM for 6 h, mitochondrial coupling ratios and the rate of ATP synthesis were not significantly different from those in fresh hearts. In conclusion, the present study has shown that long-term hypothermic storage significantly impaired both paced-evoked responses and mitochondrial function. Inclusion of BDM in the perfusion buffer during storage significantly ameliorated some of these changes.     

Effect of delayed blood processing on the yield of factor VIII in cryoprecipitate and factor VIII concentrate

Current standards for the preparation of factor VIII (FVIII) concentrates from human plasma recommend separation of plasma from red cells (RBCs) within 6 hours of blood donation, thereby reducing the volume of plasma from donated whole blood available for processing to FVIII concentrate. The decay of FVIII clotting activity (FVIII:C) in whole blood and plasma stored at 22 and 4 degrees C and the recovery of FVIII:C in cryoprecipitate and FVIII concentrate prepared from plasma separated from whole blood stored overnight at 4 degrees C were investigated. In whole blood stored at 22 degrees C and plasma stored at either 4 or 22 degrees C, 90 percent of the original FVIII:C was present at 6 hours, 80 percent at 12 hours, and 65 to 70 percent at 18 hours. At these times lower levels of FVIII:C were recovered from whole blood stored at 4 degrees C, that is, 84, 68, and 56 percent, respectively. In cryoprecipitates prepared from plasma separated from RBCs after 18 hours' storage at 4 degrees C (18-hour plasma), 43 percent of FVIII:C activity was recovered, as compared with 61 percent recovered from standard plasma separated within 6 hours of donation (6-hour plasma), p less than 0.05. With large-scale preparation of FVIII concentrates, however, the yield of FVIII:C was similar whether 18- or 6-hour plasma was used. Thus FVIII concentrates--but not cryoprecipitates--can be prepared from plasma separated from whole blood stored at 4 degrees C for up to 18 hours without undue loss of potency.     

Apoptotic markers are increased in platelets stored at 37 degrees C

BACKGROUND: PLTs for transfusion lose viability during storage in blood banking. This loss of viability is accelerated at 37 degrees C, as is the risk of bacterial contamination, and has led to the selection of 22 degrees C as the routine storage temperature. Because PLTs contain an intact apoptotic mechanism, we sought to determine whether PLTs undergo apoptosis during storage and whether storage at 37 degrees C accelerated this process. STUDY DESIGN AND METHODS: PLT-rich plasma from PLT concentrates was stored at 37 or 22 degrees C in small aliquots or whole bags, with and without cell-permeable caspase inhibitors. Number of PLTs, pH, LDH level, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium activity were analyzed over time. PLT lysates were prepared and tested for the presence and activation of apoptotic proteins by enzyme assay and Western blotting. RESULTS: PLT viability was greatly reduced after 1 to 2 days of storage at 37 degrees C; however, signs of apoptosis were evident by 3 hours after temperature shift. In temperature-stressed PLTs only, a gradual rise in caspase-3 activity was detected that correlated with the appearance of the 17- to 20-kDa cleavage products of caspase-3. Gelsolin, a caspase-3 substrate, underwent cleavage within the same time frame. Bcl-xL and caspase-2 also declined significantly; caspase-9 activity rose. Specific caspase inhibitors could prevent caspase activation but did not improve PLT cellular viability at 37 degrees C. CONCLUSIONS: PLTs contain apoptotic proteins that are activated during PLT storage at 37 degrees C and may account for the rapid decline in PLT cellular viability. Although ineffective here, inhibition of PLT apoptosis may improve PLT cellular viability.     

Sterility of plastic tubing welds in components stored at room temperature

BACKGROUND: The ability of a sterile connecting device to maintain sterility when being used to weld tubing of a blood component to be stored at room temperature, such as a platelet unit, has not been adequately documented, nor has it been determined when the tubing to be welded is filled with liquid. STUDY DESIGN AND METHODS: The sterility of sterile connecting device welds of polyvinylchloride tubing were challenged after intentional contamination of the exterior of the tubing with both gram-positive and gram-negative organisms (4 × 10(4) to 3 × 10(6) colony-forming units/mL). Welding (n = 244) was performed with the contaminated area either being wet or having been allowed to dry. At the time of the welding, the tubing segments were either empty or filled with liquid (either aliquots of white cell-reduced apheresis platelets or bacteriologic growth medium). After the welding, the liquid was passed across the weld and held in the attached transfer pack for 5 to 7 days at room temperature. RESULTS: Two welds were found to be incomplete and leaky, and both of the units involved had positive cultures. One transfer pack had inadvertently been contaminated at the time of its initial, postweld culture by a bacterium other than the one used in the experiment. Aside from these three nonevaluable units, all of the welds were sterile when cultured after the packs were held for 5 to 7 days. CONCLUSION: This study documents the ability of the sterile connecting device to maintain a closed system in the welding of blood component units to be maintained at room temperature. All welds should be closely inspected at the time of completion to detect leaks that may lead to contamination.     

The stability of angiotensin I formed at room temperature in the presence of ethylenediaminetetraacetate to subsequent incubation at 37 degrees C

It has been reported that plasma renin activity values obtained from samples containing ethylenediaminetetraacetate (EDTA) and which have been allowed to remain at room temperature for 24-48 hours prior to incubation and assay are spuriously low. Since plasma renin activity is often measured in specialised centres, considerable time delays in sample transit may be difficult to avoid. We, therefore, investigated these findings using plasma samples kept at room temperature for varying times and subsequently incubated at 37 degrees C. From these studies we conclude that plasma renin activity is not significantly affected by pre-incubation at room temperature for periods up to 48 hours. However, the precision of the assay is likely to be poorer in specimens pre-incubated at room temperature. These results are supported by the finding that there is no loss of angiotensin I upon incubation and assay of samples containing pepstatin A.     

Plasma and cryoprecipitate manufactured from whole blood held overnight at room temperature meet quality standards

BACKGROUND: With buffy coat (BC) processing of whole blood (WB) donations, the preparation of plasma occurs within 24 hours rather than 8 hours of collection. The effect of this change on coagulation factor function in plasma and cryoprecipitate was evaluated during the validation of this production method and with routine production. STUDY DESIGN AND METHODS: Plasma frozen after an overnight hold of WB was prepared via BC or whole blood filtration (WBF) methods and quality control (QC) variables were measured. Additionally, plasma prepared with the BC method was compared to plasma produced using the platelet‐rich plasma (PRP) method with an extended plasma factor analysis. Selected plasma factor levels were also measured in both cryoprecipitate and cryosupernatant plasma prepared using the WBF method from plasma frozen on the day of collection or after an overnight hold of WB. RESULTS: When comparing BC plasma to PRP plasma, coagulation factors (F)II, VII, VIII, IX, X, and XI had somewhat lower levels, and fibrinogen and antithrombin levels were elevated. As expected the most sensitive to the prolongation of production time was FVIII with 72 and 78% of the activity of PRP plasma and cryoprecipitate, respectively. However, both still met QC standards. Similarly, products made in routine production show acceptable levels of FVIII. CONCLUSION: Plasma and cryoprecipitate products, prepared using methods in which the plasma is frozen close to 24 hours after collection, meet current quality standards. The longer WB storage time has been implemented into general use in Canada.     

Stability of imipenem in Mueller-Hinton agar stored at 4 degrees C.

The purpose of the present study was to measure the stability of imipenem in Mueller-Hinton agar stored at 4 degrees C over time. MICs for Staphylococcus aureus ATCC 25923, Streptococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 were determined in triplicate daily for up to 15 days. The calculated mean time to observe a shift of one dilution in MIC endpoints was 4.33 +/- 1.25 days. For routine work, imipenem agar dilution plates should be prepared within 48 to 72 h of the test.     

Modulation of fibrinogen content in cryoprecipitate by temperature manipulation during plasma processing

In routine blood bank production of single-donation cryoprecipitate, the introduction of a 16-hour hold at 4 degrees C, with the frozen plasma units packed into polystyrene containers, resulted in plasma prethaw temperatures of -4 degrees C to -8 degrees C. This in turn resulted in cryoprecipitate fibrinogen levels that were 214 percent of those obtained when units were thawed immediately after removal from -30 degrees C storage. In scale-model production of factor VIII concentrate, plasma warmed from -30 to -10 to -15 degrees C over 18 hours before pooling and thawing yielded cryoprecipitate fibrinogen levels that were 66 percent of those found in plasma warmed to -2 to -5 degrees C over the same period. Processing -30 degrees C plasma without a warming period led to cryoprecipitate fibrinogen levels that were 40 percent of those obtained from plasma warmed to -2 to -5 degrees C. These differences were accentuated after purification of the cryoprecipitates to an intermediate-purity factor VIII concentrate. These results suggest that simple modifications in production methods allow the fibrinogen content of cryoprecipitate to be tailored to specific uses.     

Platelet concentrates: sterility of 400 single units stored at room temperature

Four hundred single platelet concentrates were prepared and stored at room temperature for 72 to 96 hours. Triple cultures of each concentrate at varying incubation temperatures revealed only four positive cultures. The Corynebacterium species and Staphylococcus species, coagulase negative, were the only organisms identified and were regarded as contaminants. No transfusion reactions of bacterial origin were observed in transfusing 10,024 other single platelet concentrates. Room temperature preparation and storage of single platelet concenrates is a safe and practical procedure.     

Increase and decrease in formic acid concentration in urine samples stored at room temperature.

Formic acid concentrations are not stable in urine samples stored at room temperature. Formic acid may increase or decrease due to bacterial contamination. Enterococci and E. coli produce formic acid under anaerobic conditions at pH values of more than 6.0. In urine sample stored at room temperature, E. coli caused a decrease in formic acid. The main substrate of formic acid formation by bacteria seems to be citric acid. Formic acid in urine is stable at room temperature for at least 7 days when concentrated acetic acid (50 microliters/10 ml) or 50 g/l thymol in isopropanol (50 microliters/10 ml) is added.     

血浆制备时间和速冻方法对冷沉淀凝血因子质量的影响

目的探讨新鲜血浆的制备时间和速冻方法对冷沉淀凝血因子质量的影响,选择合适的制备时间和速冻方法。方法随机抽取制备时间分别为2 h、4 h、6 h、8 h新鲜血浆各16人(份),采用无菌接驳机平均分为A、B两实验组,分别用平板速冻机和传统低温冰箱速冻制备新鲜冰冻血浆(FFP);在相同条件下分别对两组FFP制备冷沉淀;采用凝固法检测两组冷沉淀的凝血因子Ⅷ(FⅧ)和纤维蛋白原(Fg)。结果血浆制备时间为2 h、4 h、6 h、8 h的冷沉淀FⅧ活性(IU)A组分别为78.40±22.87、74.06±23.72、71.25±19.93、70.53±18.84,B组分别为66.60±17.12、58.08±18.19、52.57±12.26、51.19±12.51。A、B组冷沉淀FⅧ随着制备时间的延长,均呈下降趋势,相同制备时相A与B组比较均有统计学差异(P<0.05);A组FⅧ活性4个制备时相间比较差异无统计学意义(P>0.05),B组2 h与6 h、8 h比较差异均有统计学差异(P<0.05)。血浆制备时间为2 h、4 h、6 h、8 h的冷沉淀中Fg含量(mg)A组分别为114.53±24.76、117.62±27.61、114.44±22.84、120.23±26.48,B组分别为113.36±23.53、116.43±25.38、115.28±23.66、117.92±25.58,Fg含量在不同制备时间和速冻方法条件均无统计学差异。结论 8 h内制备血浆2种速冻方法均能满足冷沉淀质量要求,平板式速冻机制备血浆的冷沉淀FⅧ活性显著性高于传统低温冰箱。