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From the *Department of Anesthesiology and Intensive Care Medicine, Institute for Transfusion Medicine, Institute for Laboratory Medicine and Pathological Biochemistry, Charité-University Hospital Berlin, Charité Campus Mitte, Berlin.
Address correspondence and reprint requests to Christian von Heymann, MD, DEAA, Department of Anesthesiology and Intensive Care Medicine, Charité - University Hospital Berlin, Charité Campus Mitte, Schumannstr. 20–21, D-10117 Berlin, Germany. Address e-mail to christian.von_heymann{at}charite.de.
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Abstract |
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METHODS: Plasma of 20 healthy donors was sampled, separated, and distributed in 3 plasma bags. Within 2 h after sampling plasma bags was frozen at a temperature of –30°C to –40°C and stored for at least 8 wk. After sampling (baseline) as well as immediately and 1, 2, 4, and 6 h after thawing, the activity of FV, FVII, FVIII, fibrinogen, fibrin monomers (FM), d-dimers (DD), 
2-antiplasmin (2-AP), and protein S (PS) was determined from each plasma bag.
RESULTS: From 1 h to 6 h after thawing, no significant differences in the activity of the investigated coagulation markers dependent on the thawing procedure were found. However, immediately after thawing and independent of the thawing procedure, the activity of FVII was significantly decreased (P < 0.01), whereas FM were significantly increased (P = 0.001).
CONCLUSION: The thawing procedures studied exhibited no significant influence on activity and stability of the investigated markers of coagulation over the study period. The decreased activity of FVII and the clinical significance of the increase in FM require further research.
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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FFP is prepared within 6–8 h after collection and frozen at –30°C to –40°C according to the German guidelines for the production of blood and blood components and for the utilization of blood products (hemotherapy) (3). On the one hand, the remaining activity of coagulation factors after thawing is crucial for the efficacy of plasma replacement therapy. In this regard, previous publications have shown that, with the exception of FV (4) and FVIII (5), coagulation factors in the FFP remain stable for at least 24 h after thawing when kept at 4°C (6). On the other hand, activation of coagulation zymogens in the cold (7) may be a serious side effect which can predispose for thrombosis or disseminated intravascular coagulation (DIC).
Thawing devices must avoid damage to the plasma bags and bacterial contamination while rapidly warming blood components. In emergency situations requiring the replacement of greater volumes of FFP, the efficacy of thawing devices is primarily judged by the speed of thawing, the activity of clotting factors, and the detection of activated coagulation factors after the thawing procedure.
The objective of this study was to analyze the influence of the thawing process of different commercially available devices on the activity of clotting factors, inhibitors, and activation markers of the hemostatic system. Furthermore, the activity of these factors over a time period of 6 h after thawing was measured.
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METHODS |
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The plasma units were thawed using 2 commercially available devices which are approved for these types of blood components in several European countries: a) the Plasmatherm III® (Barkey, Leopoldshöhe, Germany), which is a shaking water bath that uses pads filled with warm water to thaw blood products, and b) the Transfusio-therm 2000® microwave blood warmer (EIC Umwelt- und Medizintechnik Ltd. Heilbad Heiligenstadt, Germany). The warming temperature of the Plasmatherm III® was 37°C and the warming cycle is preset at 30 min by the manufacturer. The Transfusio-therm 2000® terminates heating at a surface temperature of 35°C, which is measured electronically with two separate thermistors (PT 100 class B; UST Umweltsensortechnik GmbH, Geschwenda, Germany).
A control series of FFP was thawed under running warm tap water at a temperature of 42°C. The plasma bags were placed in a bucket with warm water dripping over them. The warm water was allowed to flow out of the bucket into the basin. Complete liquification of the plasma was determined by manual assessment. There was no interference with the thawing process. The temperature of the tap water was measured with a calibrated electronic thermometer (Neolab®, Heidelberg, Germany) at 30°C ± 0.1°C) and controlled for at least 5 min to achieve a steady state. The 3 separated plasma bags were allocated to each of the thawing procedures studied to prevent a variation of any influence of clotting factor activity as the result of different ABO groups (8). At one time 2 bags of plasma were thawed in both devices and under running warm water, respectively. In case the plasma was not completely liquefied in the bags after the first cycle of thawing, a second cycle was performed. The plasma was thawed under running warm water until it was completely liquefied. The heating times for each of the 20 consecutive thawing sessions of each procedure were recorded.
After completion of the thawing procedure the temperature was measured by introducing a calibrated electronic thermometer (Neolab®) at 30°C ± 0.1°C) into each plasma bag. For serial measurements of clotting factors the plasma bags were kept at room temperature (25°C) for 6 h and discarded thereafter.
Plasma was taken in aliquots of 5 mL from each plasma bag for coagulation factor measurements at baseline and immediately and 1, 2, 4, and 6 h after thawing. All measurements were performed using the same assays. Measurements of clotting factors were performed immediately after sampling and used only commercially available assays. Clotting factor assays included factors V (FV), VII (FVII), and VIII (FVIII), fibrinogen, d-dimers (DD), fibrin monomers (FM), 2-antiplasmin (2-AP), and protein S (PS).
FV (reference range: 60%–150%) and FVII (reference range: 50%–150%) were measured using a specific factor-deficient plasma and coagulation was started with Innovin® (Dade Behring GmbH, Marburg, Germany). FVIII (reference range: 50%–150%) was measured using a partial activated thromboplastin, FVIII-deficient plasma and was started with 28 mmolar CaCl2.
Fibrinogen (reference range: 150–450 mg/dL) was measured by the method of Clauss, and PS (reference range: 60%–140%) by the use of a PS assay. The above named assays used clot-based endpoints. Furthermore, 2-AP (reference range: 70%–130%) was measured with a chromogenic assay by photometric absorbance (405 nm). The above-named factors and inhibitors were assayed on the STA® coagulation analyzer.
The d-dimer assay (reference range: <0.5 mg/L) used a latex-enhanced immunoassay (LIA) with turbidimetric determination. Plasma samples for the FM-assays were frozen and stored at –80°C until en bloc-determination. The FM-assay used the reagent Berichrom® FM and was measured by photometric absorbance (405 nm over 60 s) on a BCT® Analyzer (Dade Behring GmbH). The lower detection limit was <1 mg/L and the reference range of the FM-assay was: <15 mg/L.
Data were analyzed using the Statistical Package for Social Sciences for Windows, version 11.0 (SPSS, Inc., Chicago, IL) licensed for Charité University Hospital. Results are given as median and interquartile range (IQR), as it was assumed that data were not normally distributed because of the small sample size. The clotting factor activity between the different thawing procedures was analyzed using the Kruskal-Wallis test. The clotting factor activity between the time points studied was assessed with the Friedman test globally and the Wilcoxon’s ranked sum test locally. In addition, a nonparametric multivariate analysis of variance (MANOVA) for repeated measurements, longitudinal data, and small sample sizes in a two-factorial design (first factor (group): different thawing procedures, second factor (time) (SAS version 8 macro F1_LD_F1; SAS Institute, Cary, NC) (9) was performed. This analysis compared 4 measurements (from 1 h to 6 h after thawing) simultaneously on the corresponding response curves. A P < 0.05 was considered statistically significant.
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RESULTS |
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The heating times of the thawing procedures (Table 2) were significantly different (P = 0.01), with the shortest thawing time for the running warm water procedure (median, 5 min; IQR, 5–6 min.). The Transfusio-therm 2000® took 6 min (6–7.75 min) to liquefy the plasma. The Plasmatherm III® thawed all plasma samples within the preset time of 30 min (1 thawing cycle). The temperature of the plasma immediately after the thawing procedure was significantly different between the methods/devices used (Table 2) ranging from 25.5°C for the Transfusio-therm 2000® to 29.0°C for the Plasmatherm III®.
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Table 3 shows the activity of clotting factors, inhibitors, and activation markers at baseline and immediately after thawing. Except of FVIII all parameters were within the normal range. Compared with baseline, FVII activity was significantly lower (P < 0.01), whereas fibrin monomers that were below the lower detection limit at baseline showed a significant increase (P = 0.001) after thawing. The statistical analysis showed that these results were not significantly different between the thawing procedure (Table 3), i.e., the decrease in FVII activity and the increase in FM were independent of the thawing procedures. Fibrinogen, FV, FVIII, d-dimers, 2-AP, and PS were not significantly different to baseline values.
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With regard of the time period from 1 h to 6 h after thawing the MANOVA showed that the thawing procedures studied had no significant impact on the activity of clotting factors and coagulation parameters (Fig. 1).
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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2-AP, and PS remained unchanged compared to baseline. These results were independent of the thawing procedure. Secondly, the activity of clotting factors, inhibitors and activation markers of the hemostatic system were not significantly influenced by the thawing procedure between 1 hour and 6 hours after thawing. Independent of the thawing procedure, we found a significant decrease of FVII activity that is supported by previous results describing a slight decrease of FVII activity in FFP frozen within 24 hours after collection (10). A decrease in FVII activity has been contributed to the surface of the plasma bags (7), which were standardized in our study. Furthermore, the influence of the thawing procedures seems to be negligible as the decrease in FVII-activity was shown to be the same for all three methods (Table 3). The median FVII activity immediately after thawing remained above 100% for all procedures, which attenuates the significance of this result for the clinical setting. However, recent studies did not describe significant changes in FVII activity after repeated thawing and freezing of FFP (10,11) or in whole blood (5), which requires further investigation to elucidate the mechanism behind the decrease in FVII activity.
FM, a marker of early fibrin formation and activation of the clotting system, were detected immediately after thawing, whereas these were below the lower detection limit at baseline. A recent study detected fibrin monomers in FFP that were not induced by whole-blood filtration (12). It may be hypothesized that fibrin monomer formation was caused by a freezing-induced generation of thrombin, resulting in an increased turnover of fibrinogen with the cleavage of FM. Unfortunately, we did not measure thrombin-antithrombin-complexes to assess thrombin generation specifically. However, according to our results the thawing procedure itself had no influence on the generation of fibrin monomers. Previously the detection of fibrinopeptide A in FFP dependent on the donation time (13) was described, which also indicates that a thrombin-induced fibrin formation takes place during the manufacturing process (sampling, freezing, storage or thawing) of FFP. This activation of the hemostatic system already during the sampling of plasma is not supported by our results, as FM were not detected and d-dimers were within the normal range at baseline. Whether the freezing or thawing process of the FFP induces thrombin generation resulting in some degree of hemostatic activation can not be excluded by our results, but requires further investigations.
Regarding FV and FVIII, which are considered relatively labile, our results confirm data of Dzik et al. (4) and recent data (14) that indicate no significant differences in FV and FVIII immediately after first-time thawing but a significant decrease after second-time freezing. In contrast, FVIII was significantly decreased when plasma was frozen at a temperature of –80°C (15) or within 24 hours after collection (10) instead of 6–8 hours (3,12), emphasizing the temperature-dependency of FVIII. This may be supported by data from Akerblom et al. (16), who showed a significant decrease of FVIII activity immediately after thawing that was dependent on the speed of the freezing procedure. The drop in FVIII activity immediately after thawing was significant only for the rapid freezing procedure. In this study a drop in FV activity to 92% of pre-freezing levels was measured, which confirms some degree of freezing-associated instability of FV. In contrast, Nifong and coworkers (10) measured that FV increased by 8.5% in the first measurement after thawing, which confirms the statistically insignificant increase of FV in our results.
In our results, d-dimers, a marker of early fibrinolysis, were within the normal range immediately after thawing, which is in accordance with recent results (12). Immediately after thawing fibrinogen, 2-AP (12) and PS (5,12) were not different from baseline, confirming the stability of these clotting factors and inhibitors in FFP. The insignificant changes of clotting factors and inhibitors applied to the two approved thawing devices studied and to the experimental running warm water procedure. The running warm water procedure used a significantly higher thawing temperature (Table 2), which underscores the relative stability of the clotting factors and inhibitors studied. Immediately after thawing, the temperature within the plasma bags was significantly different between procedures (Table 2), but this did not exert a significant impact on clotting factor and inhibitor activity. This may be explained by the still relatively low thawing temperatures (maximum: 42°C for the running warm water procedure), which probably did not result in a significant degradation of clotting factors.
In a second step we analyzed the impact of the thawing procedures on the activity of clotting factors and inhibitors over a time period of 6 hours after thawing. Our results found no significant differences between procedures over time. Sohngen et al. (17) compared a microwave to a shaking water bath. For the microwave the change in FVIII and FIX activity was significantly less compared to the waterbath. But these results were found in FFP with a high cell contamination, whereas no differences were found in FFP with low cell contamination. We assume that the standardized cell-separation performed in our investigation yielded the previously described low cell contamination and prevented significant changes in cell count. This may have influenced our results. Furthermore, the microwave oven studied by Sohngen et al. represented an early generation of microwaves that have raised concerns regarding the reported overheating of blood products (18,19). The present study evaluated the Transfusio-therm 2000®, a new microwave that, according to a recent report (20), did not lead to overheating of plasma and the surface of the plasma bags. Therefore, a local overheating which may have resulted in denaturation of clotting factors or inhibitors seems unlikely, which is confirmed by our results. For the time period of 1 to 6 hours after thawing, no significant differences in clotting factor or inhibitor activity between the shaking water bath (Plasmatherm III®) and the microwave were found. Likewise, no thawing procedure studied significantly influenced the levels of FM, d-dimers, or 2-AP. It may be concluded that fibrin formation and early fibrinolysis were not affected by the approved thawing devices. The experimental procedure of running warm water at a significantly higher temperature of 42°C yielded no significant differences in clotting factor or inhibitor activity. This may give support to the assumption that a higher thawing temperature may be safely applied to thaw FFP without a significant reduction in clotting factor or inhibitor activity. For clinical situations requiring a rapid and large replacement of FFP, this seems to be of special interest, as thawing times may be substantially reduced.
We conclude that, first, the thawing procedures applied had no significant influence on the activity of clotting factors, inhibitors, and activation markers of the hemostatic system in FFP. The decrease of FVII immediately after thawing and, in particular, the clinical significance of FM, detected independent of the thawing procedure, requires further investigation. Second, furthermore the results of our investigation indicate that a thawing temperature of 42°C did not significantly reduce the activity of clotting factor and inhibitors within 6 hours after thawing, which supports data from the literature (4,10,14). This may allow a faster thawing process and allocation of FFP in massive transfusion.
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Footnotes |
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Accepted for publication June 2, 2006.
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