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Indicators of erythrocyte damage after microwave warming of packed red blood cells.

Transfusions of red blood cells are necessary to increase the oxygen-carrying capacity in patients after major trauma or during and after surgery. Units of packed red blood cells (PRBCs)3 are generally stored at 4 [+ or -] 2[degrees]C according to German regulations (1). Cold storage is considered to be interrupted if the temperature of the blood increases above 8[degrees]C. The US Food and Drug Administration regulation recommends 1-6[degrees]C (2). For patients requiring large volumes of PRBCs for transfusion, units must be warmed immediately before transfusion to prevent hypothermia. Especially in emergency situations requiring massive transfusions, this need for warming can lead to considerable delay.

The use of microwaves to warm blood products was propagated soon after the introduction of microwave ovens for other purposes in the mid-1950s and came into regular use in the 1970s. The devices provided shorter heating times than did conventional heating in a water bath, but several reports of complications from overheating of blood products (3-5) led to widespread abolishment of microwave blood warmers. Considerable debate remains regarding the use of these devices.

The primary concern with microwave blood warmers is the development of hemolysis as a result of general overheating and local hot spots within the blood units. Several studies have evaluated the concentrations of hemolysis markers, such as free hemoglobin (FHb) and lactate dehydrogenase (LDH) in PRBCs and whole blood after microwave heating (4-7). Recent evaluations showed no clinically relevant difference between blood heated by microwave blood warmers and blood heated by other devices with respect to these markers (6, 7). Consequently, the use of microwave blood warmers is currently increasing.

One shortcoming of these studies is that either hemolysis was measured immediately after heat exposure or the authors did not provide the time of measurement. One experimental study with chicken erythrocytes, however, demonstrated a substantial delayed hemolysis after heat exposure (8). Heating to 51.5[degrees]C for 180 min caused 13% hemolysis measured immediately after heating but almost 100% hemolysis measured after 24 h.

Furthermore, global markers are of limited value for the detection of hot spots as a result of localized overheating in a microwave oven. It has been shown that characteristic changes exist in overheated blood with respect to cell size and hemoglobin distribution (9). These changes are similar to the changes in the blood of patients with severe burns (10, 11). The increasing availability of flow cytometry has made this method a powerful and rapid means to examine changes in large numbers of individual cells. To our knowledge, there have been no previous studies describing cellular changes of erythrocytes after warming of PRBCs.

Material and Methods

Samples of PRBCs were warmed to different temperatures from room temperature to 57[degrees]C in a water bath. Room temperature was chosen as the control value because PRBCs that are not actively warmed will, in clinical practice, be transfused after reaching ambient temperature to avoid discomfort and hypothermia of the patient. The concentrations of FHb and the isoenzymes LDH1 and LDH2 [also known as [alpha]-hydroxybutyrate dehydrogenase (HBDH)] were measured immediately after heating and after 48 h. Flow cytometry was used to characterize cells and to evaluate changes in erythrocyte antigen patterns ([Na.sup.+]-[K.sup.+]-ATPase, [Ca.sup.2+]-ATPase, and spectrin) with respect to thermal changes. After we established a detection assay for general and localized overheating, we performed measurements before and after the heating of PRBCs with a commercially available microwave blood warmer that is in regular use in our department.

PRBCs that had been discarded by the blood bank of our institution because of age or undocumented interruption of cool storage were used for the experiments. The units were stored in a refrigerator certified for the storage of PRBCs before and between the measurements. Before the experiments, the units were checked visually for signs of clot formation or hemolysis. Samples were heated by immersing 10-mL samples in closed plastic tubes in a calibrated water bath (Haake DC 10; Haake GmbH) for 60 min. The temperature of the samples was cross-checked with a calibrated thermometer (Testo[R] 110; Testo GmbH & Co; accuracy [+ or -] 0.2[degrees]C).

MARKERS OF OVERHEATING

Samples from 12 units were heated to room temperature (~20[degrees]C), 37, 42, 47, 52, and 57[degrees]C. Samples for the measurement of hemolysis markers (FHb and HBDH) and for the characterization of cell changes by flow cytometry were taken from the same plastic tubes.

HEMOLYSIS MARKERS

For preparation of cell-free supernatant, an aliquot of the PRBCs was centrifuged at 3000g for 10 min. We measured [alpha]-HBDH activity in the supernatant according to the recommendation of the German Society for Clinical Chemistry (12), using a commercially available assay (Roche Diagnostics) on a Hitachi 917 clinical chemistry analyzer (Hitachi Ltd.). FHb was measured by the formation of cyanhemoglobin (13), using a commercially available assay (Roche Diagnostics) on a Hitachi 917 analyzer.

Because the addition of lidocaine had led to substantial changes in the heat-induced concentration changes of the hemolysis markers in previous measurements (data not shown), these measurements were performed without lidocaine.

FLOW CYTOMETRY MEASUREMENTS

For flow cytometry measurements, we used a Becton Dickinson FACSCalibur[TM] flow cytometer that was calibrated using fluorescein-labeled chicken erythrocytes. For each measurement, 15 000 cells (events) were evaluated. In preliminary measurements, two regions of interest of the forward-to-sideward side scatter (FSC/SSC) ratio were defined (Fig. 2). In region 1 (R1), a large proportion of erythrocytes could be found at room temperature. Without heat exposure, the cells in this region could not be labeled with the fluorescent antibodies. In region 2 (R2), a substantial number of cells could be found after heat treatment ([greater than or equal to] 52[degrees]C); the cells in this region showed fluorescence after tagging with fluorescein. The analysis of cell changes was performed using these regions ("gating"). The gate settings were not changed during the experiments. To compare measurements we used the percentage of the gated events that could be assigned to these regions (%) and the median of the fluorescence.

For flow cytometry analysis, the PRBCs were diluted 1:1000 in 1x phosphate-buffered saline. The cells were washed three times by centrifugation at 9000g for 10 min and resuspension in phosphate-buffered saline. The detection antibodies were then added, and the cells were incubated for 60 min at room temperature. The primary antibodies were mouse monoclonal anti-human [Ca.sup.2+]-ATPase IgG (Maine Biotechnology Inc.; MAP 457P), mouse anti-human [Na.sup.+]-[K.sup.+]-ATPase ([alpha]-subunit) IgG (cat. no. A-276; Sigma-Aldrich), and mouse monoclonal antispectrin ([alpha] & [beta]) IgG (cat. no. S3396; Sigma-Aldrich).

After three washing steps, the cells were incubated for 60 min with a fluorescein-conjugated goat anti-mouse IgG F(ab') antibody (Rockland Inc.) in a dark environment. The cells were then washed, and flow cytometry was performed. Measurements were generally done in parallel with room temperature samples as a negative and blood heated to 52[degrees]C as a positive control.

Previous experiments had shown a higher sensitivity of erythrocytes to heat challenge with changes at temperatures <47[degrees]C after incubation with lidocaine (data not shown). We therefore repeated the flow cytometry measurements after adding 200 g/L lidocaine to the samples (final concentration of lidocaine, 15 mmol/L). Gating for these experiments was the same as described above.

SENSITIVITY FOR LOCAL HOT SPOTS

We examined the sensitivity of flow cytometry and the assays for markers of hemolysis to detect local hot spots by heating samples from the 12 PRBC units to 52[degrees]C. We then diluted the heated blood stepwise (1:1 to 1:64) in blood stored at room temperature from the same unit. In a 320-mL PRBC unit, a local hot spot of 1:64 of the unit would equal ~5 mL. Laboratory and flow cytometry measurements were performed as described above. Because the results for the different antibodies had been very similar, these experiments were performed with the anti-spectrin antibody only.

EVALUATION OF MICROWAVE HEATING

The transfusio-therm[R] 2000 microwave blood warmer (Zeipel Medical GmbH) is currently being used in several hospitals in Europe and Japan. The device has holders for three bags of fresh-frozen plasma or PRBCs. The holders are equipped with metal shields to protect the thinner peripheral regions of the blood units from excess doses of microwave radiation. During the heating process, the holders rotate continuously within the device. Two separate temperature probes with separate regulatory circuits are installed in each holder.

We used 30 units of PRBCs for these experiments. Before heating, the units were gently mixed and opened at the filling tubes. After mixing, 10 mL of blood was removed from all units for control measurements. Lidocaine (200 g/L) was injected into 15 of the units to a final concentration of 15 mmol/L. The bags were then sealed with an electrical welder. The units were heated until the cutoff temperature of the device at 35[degrees]C. For comparisons, samples from each unit without lidocaine were left at room temperature and heated to 52[degrees]C in a water bath as described above. Laboratory and flow cytometry measurements were then performed as described above. Samples from the units with lidocaine were heated to 37 and 47[degrees]C in a water bath; for these samples, only flow cytometry measurements were performed.

STATISTICAL ANALYSIS

The data are generally expressed as the mean (SD). Statistical analysis was performed with the software package SPSS 10.0[R] (SPSS GmbH) on a Windows[R] PC. Gaussian distribution was tested using the Kolmogorov-Smirnov goodness-of-fit test. All data sets were compared with the t-test for paired samples. P <0.05 was considered statistically significant. Because some of the datasets used for correlation analysis did not follow a gaussian distribution, correlation analysis was performed with the Spearman rank correlation. Curve fitting was performed with the curve-fitting algorithms provided in the SPSS software package.

Results

MARKERS OF OVERHEATING

We evaluated 12 PRBC units with a mean (SD) hematocrit of 55.9 (4.9)%, hemoglobin of 174 (16) g/L, and red cell count of 6.0 (0.5) x [10.sup.6]/[micro]L. The mean (SD) age of the units at the time of measurement was 39.3 (9.8) days.

Hemolysis markers. FHb and the HBDH values were higher than the reference values in humans [FHb, <0.3 g/L; HBDH, <140 U/L (14)] in the room temperature samples. FHb and HBDH showed a parallel and exponential increase with heat (see Fig. 1 and Table 1 of the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol49/ issue5/).

[FIGURE 1 OMITTED]

Both FHb and HBDH increased further 48 h after warming to room temperature (P < 0.001 compared with measurements immediately after heating for all temperatures). After heating to 37[degrees]C, the proportion of delayed increase in FHb and HBDH concentrations was similar to that after warming to room temperature. In the samples that were heated to 42[degrees]C, the FHb concentrations were more than twice as high as the concentrations measured immediately after heating, whereas the HBDH was increased ~1.5-fold. Forty-eight hours after heating to 47[degrees]C, there was a threefold increase compared with the measurements immediately after heating for both FHb and HBDH.

Flow cytometry measurements. In preliminary measurements, two regions of the FSC/SSC ratio were defined and were used throughout the measurements without changes (see Fig. 2). At room temperature, there was only spontaneous fluorescence in R1 in all 12 samples. This was the case for [Ca.sup.2+]-ATPase, [Na.sup.+]-[K.sup.+]-ATPase, and spectrin antibodies [see Table 2 of the online Data Supplement (available at http://www.clinchem.org/content/ vol49/issue5/)]. A large proportion of the 15 000 counted erythrocytes could be found in R1 (Fig. 2A). The remainder of the erythrocytes showed no specific pattern of distribution of the FSC/SSC ratio. We found only a very low percentage of events in R2. This pattern was identical for the samples that had been stored at 37[degrees]C. Above 42[degrees]C, we observed significant fluorescence in R1 with all three antibodies (Fig. 3 and Table 2 of the online Data Supplement). The pattern of FSC/SSC distribution was significantly changed after heating to 47[degrees]C and above (Fig. 2B and Table 2 of the online Data Supplement).

[FIGURE 2 OMITTED]

In a second step, we added 15 mmol/L lidocaine to samples from the same 12 PRBC units. After heating to 37[degrees]C, the median fluorescence was increased and the proportion of erythrocytes in R1 was decreased [Table 3 of the online Data Supplement (available at http://www. clinchem.org/content/vol49/issue5/)] compared with the samples without lidocaine. A very low proportion of the cells were found in R2. After heating to 42[degrees]C, we observed a trend toward a higher median fluorescence in R1, which was statistically significant for [Ca.sup.2+]-ATPase and [Na.sup.+]-[K.sup.+]-ATPase antibodies. There were also substantially more events in R2 than at 37[degrees]C. After heating to 47[degrees]C, we found a significant increase in the median fluorescence in R1 with all antibodies. The proportion of cells in R1 was significantly lower, and the proportion of the events in R2 increased significantly.

[FIGURE 3 OMITTED]

To further evaluate the changes with increasing temperature, we heated samples to room temperature, 37, 39, 41, 43, 45, and 47[degrees]C after the addition of 15 mmol/L lidocaine. The erythrocytes were labeled with the anti-spectrin antibody [Table 4 of the online Data Supplement (available at http://www.clinchem.org/content/vol49/ issue5/)]. We observed a gradual increase in the median fluorescence in R1. Compared with the values at room temperature, the differences were statistically significant at temperatures >43[degrees]C. At the same time there was a stepwise increase of the proportion of events in R2 with statistically significant differences at temperatures >43[degrees]C.

SENSITIVITY FOR LOCAL HOT SPOTS

Hemolysis markers. Blood from 12 PRBC units was heated to room temperature and to 52[degrees]C. The blood heated to 52[degrees]C was then diluted stepwise (1:1 to 1:64) in the blood warmed to room temperature. The concentrations of FHb and HBDH were measured at room temperature, at 52[degrees]C, and in the dilutions 1:8, 1:32 and 1:64 [Table 5 of the online Data Supplement (available at http://www.clinchem. org/content/vol49/issue5/)]. Even at the 1:64 dilution, both FHb and HBDH were still significantly increased compared with the room temperature samples. At this dilution, the FHb and HBDH concentrations in some of the samples were in the range of some of the room temperature samples. We observed a stepwise decrease in hemolysis markers with significant correlations between the dilution factor and both FHb (r = 0.41; P = 0.001; n = 60) and HBDH (r = 0.91; P <0.001; n = 60).

Flow cytometry measurements. Samples diluted 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 were labeled with anti-spectrin antibodies. The median fluorescence was significantly increased in R1 in the samples that had been heated to 52[degrees]C [Table 6 of the online Data Supplement (available at http://www.clinchem.org/content/vol49/issue5/)]. The proportion of events in R1 and R2 was significantly different after heating to 52[degrees]C compared with room temperature. The number of events in R2 decreased in parallel with the dilution factor. At a dilution of 1:64, there was still a significant difference compared with the samples that were stored at room temperature, but the absolute number of events in some of the samples was within the same range as in some of the room temperature samples.

The dilution factor and the median fluorescence were significantly correlated (R1, r = 0.62; P <0.001; n = 96; R2, r = -0.31; P = 0.002; n = 96), as were the dilution factor and the percentage of events in the regions (R1, r = -0.61; P <0.001; n = 96; R2, r = 0.97; P <0.001; n = 96). For R2, the relationship between the dilution factor and the percentage of events could be described by a quadratic function (Fig. 4).

EVALUATION OF MICROWAVE HEATING

FHb and HBDH. Twelve units of PRBCs [mean (SD) age, 32.5 (8.3) days; weight, 343.8 (29.2) g] were used. Three more units with substantial hemolysis by visual inspection [age, 36.3 (4.0) days; weight, 336.3 (18.0) g] were evaluated separately. No unit showed signs of overheating on visual examination after microwave warming.

At room temperature, the mean (SD) FHb was 0.5 (0.5) g/L (minimum-maximum, 0.03-1.39 g/L). After warming to 35[degrees]C with the microwave blood warmer, FHb was 0.5 (0.6) g/L (0.04-1.99 g/L). The difference between the groups was not statistically significant. The three hemolytic samples had a mean (SD) FHb of 3.3 (2.7) g/L (1.63-6.36 g/L); after heating to 35[degrees]C, the mean FHb was not significantly different: 2.6 (1.5) g/L (1.69-4.27 g/L).

[FIGURE 4 OMITTED]

Samples warmed to room temperature had a mean (SD) HBDH concentration of 531 (358) U/L (minimum-maximum, 106-1040 U/L). After heating to 35[degrees]C with the microwave device, the mean (SD) concentration was 526 (384) U/L (135-1427 U/L) with no statistically significant difference. The three hemolytic units had a mean (SD) HBDH concentration of 2585 (1797) U/L (1496-4660 U/L) with no significant difference after heating to 35[degrees]C [2160 (1107) U/L; 1270-3400 U/L].

Flow cytometry. We compared the percentage of events in R2 and the median fluorescence in R1 by flow cytometry.

In samples without lidocaine, at room temperature, 0.2 (0.04)% (minimum-maximum, 0.11-0.23%) of the cells in the 12 PRBC units that did not have visual evidence of hemolysis were in R2; the median (SD) fluorescence in R1 was 126.1 (28.5), with a minimum of 105 and a maximum of 187. In the three hemolytic units, 0.1 (0.04)% of the events were in R2 (minimum-maximum, 0.11-0.19%). The median (SD) fluorescence in R1 was 139.3 (40.5), with a minimum of 113 and a maximum of 186.

After heating to 52[degrees]C in a water bath, the percentage of events in R2 increased to 14.8 (6.4)% (1.28-25.21%; P <0.001). The median (SD) fluorescence in R1 was also increased [189.1 (27.8); minimum-maximum, 156-236]. The three hemolytic samples had a higher percentage of events in R2 [22.8 (5.8)%; 17.88-29.14%] and a higher median (SD) fluorescence in R1 [211.3 (14.6); minimum-maximum, 195-223] than the other samples.

After heating to 35[degrees]C with the microwave blood warmer, there were no significant changes in the mean (SD) percentage of events in R2 [0.2 (0.05)%; minimum-maximum, 0.08-0.25%] or the median (SD) fluorescence in R1 [124.6 (29.6); minimum-maximum, 85-184]. The three hemolytic units had a higher number of events [0.2 (0.1)%; 0.16-0.32%] and a higher median fluorescence [147.3 (30.1); 128-182] than the remainder of the samples.

The portions of the 15 PRBC units to which 15 mmol/L lidocaine had been added had a mean of 0.2 (0.5)% events (minimum-maximum, 0.03-1.95%) in R2 at room temperature. The median fluorescence in R1 was 105.0 (4.4), with a minimum of 96 and a maximum of 114.

Heating to 47[degrees]C in a water bath produced significant increases in the number of events [33.9 (7.7)%; minimum-maximum, 20.51-44.51%; P <0.001] and in the median fluorescence [143.7 (17.1); minimum-maximum, 114-181; P <0.05].

After samples were heated to 37[degrees]C in a water bath, there was no significant increase in events [0.1 (0.1)%; minimum-maximum, 0.04-0.29%] or median fluorescence [107.3 (4.3); minimum-maximum, 100-115] compared with the room temperature samples.

We found no significant differences between the room temperature samples, the samples that had been heated to 37[degrees]C in a water bath, and the samples from the units heated to 35[degrees]C in the microwave device. This was true for the number of events [35[degrees]C, 0.1 (0.2)%; minimum-maximum, 0.02-0.93%] and the median fluorescence [35[degrees]C, 106.4 (4.7); minimum-maximum, 100-117].

Discussion

The primary results of our study were as follows: (a) Analysis of the FSC/SSC ratio and fluorescence labeling with anti-spectrin antibodies can be used to identify damage to individual erythrocytes after heating to >52[degrees]C. (b) The detection threshold of this method can be lowered to 47[degrees]C by the addition of 15 mmol/L lidocaine to the samples. (c) Analysis of FHb and HBDH can detect heat damage to PRBC units in the same temperature range. If PRBCs are heated to temperatures of 42 and 47[degrees]C, a considerable part of the hemolysis occurs after 48 h. (d) Both methods can detect the localized overheating of ~5 mL in a 320-mL PRBC unit. (e) We did not observe damage to PRBCs or an increase in hemolysis after heating to 35[degrees]C with a commercially available microwave blood warmer.

We observed labeling of cell membrane antigens with all antibodies only after exposure to temperatures >42[degrees]C. There are several possible explanations for this phenomenon. Destruction of the cell membrane may lead to an influx of antibodies to the inner side of the cell membrane and allow them to bind to previously inaccessible sites. Permeability changes of the cell membrane could also explain the observed changes in FSC, which is correlated to cell size. It has been shown previously that the membranes of erythrocytes make three transitions after heating to 50.0, 56.8, and 63.8[degrees]C, respectively (15). Both spectrin and the antibody-binding site of the [alpha]-subunit of [Na.sup.+]-[K.sup.+]-ATPase (16, 17) are on the cytoplasmic side of the cell membrane, and the anti-[Ca.sup.2+]-ATPase antibody we used is also reported to bind to the [Ca.sup.2+] translocating channel protein associated with the intracellular membrane [Ca.sup.2+]-[Mg.sup.2+]-ATPase (18), which would support this hypothesis. Another mechanism of action could be heat-induced denaturation of the detected proteins themselves, leading to a change in the tertiary structure of the proteins with subsequent exposure of antigen-binding domains. This theory is supported by the fact that heat-induced changes in the properties of all three proteins can be observed in this temperature range. It has been demonstrated that the unfolding of spectrin occurs at 49.5[degrees]C (19), but the denaturation of spectrin alone seems insufficient to induce thermohemolysis (15). The activities of both [Ca.sup.2+]-ATPase and [Na.sup.+]-[K.sup.+]-ATPase are decreased after heating to 45[degrees]C (20) and 43[degrees]C (21), respectively. This could be the result of protein denaturation leading to a change in conformation and antibody binding, although it has been shown that there is no increase in Lowry-reactive protein fragments after the heating of erythrocytes up to 49[degrees]C (22).

Local anesthetics sensitize cells to hyperthermia (23, 24). It has been shown by differential scanning calorimetry that the incubation of human erythrocyte ghosts and sarcoplasmic reticulum with lidocaine leads to a decrease in the transition temperature of the transmembrane domain of the band III protein and [Ca.sup.2+]-ATPase. This produces a decrease in the denaturation temperature (25). A previous study showed a similar decrease in the critical temperature of fragmentation from 49 to 47[degrees]C with diamide, but not with several other membrane-active agents (26).

We cannot firmly conclude from our data which of these factors are

the most important contributors to the observed changes in antibody binding, but the uniformity of antibody binding to all three antigens at temperatures >47[degrees]C and the change in cell size makes it likely that a change in the permeability of the cell membrane to antibodies is an important factor. This is also supported by the results of other investigators, who found a linear increase in the potassium leakage of human erythrocytes between 46 and 54[degrees]C (27) and as well by the reports of massive changes in erythrocyte size and hemoglobin distribution after the heating of blood specimens to temperatures >49[degrees]C and in patients with severe burns (9-11).

Especially after heating to 42 and 47[degrees]C, there was a considerable further increase in hemolysis after 48 h compared with the measurements immediately after heating. The time between heating and measurement is therefore an important factor for the comparison of hemolysis after blood warming. This should be considered when new blood warming devices are being evaluated.

The results of the flow cytometry and the biochemical assays were significantly correlated with the presence of heat-damaged erythrocytes in the dilution assay. Both assays were capable of detecting heat-damaged (52[degrees]C) samples diluted in untreated samples up to a dilution of 1:64. This would correspond to the localized overheating of 5 mL in a 320-mL unit of PRBCs. At this dilution the absolute values of the hemolysis markers in some samples were similar to the room temperature values of others. The transfusion of units with a lower percentage of overheated cells would probably have little clinical relevance. The methods therefore provide an adequate means to evaluate blood-warming devices for general and localized overheating.

When we used the presented methods to test PRBCs warmed with the microwave device, neither method detected signs of overheating. On the basis of these results, the microwave system can be safely used in the present configuration.

In conclusion, we believe that this methodology can be used to detect both hot spots and general overheating in PRBC units. The sensitivity is sufficient to be useful for the future testing of blood-warming devices. Another potential application for the flow cytometry measurements may be the initial screening of burn victims to assess the risk for future complications, such as kidney failure from hemoglobinemia. The substantial amount of delayed hemolysis that we found based on the measurement of biochemical markers of hemolysis after heat exposure should be taken into account in both recommendations for the temperature set points for the warming of PRBCs and for future testing of blood-warming devices. The exponential increase in hemolysis beyond 42[degrees]C suggests that the temperature set point for blood warmers should allow a sufficient margin of safety for these temperatures.

This project was funded by the research foundation of the Department of Anesthesiology and Intensive Care Medicine at Giessen University Hospital. The foundation received an unrestricted donation from Zeipel Medical GmbH (Bad Heiligenstadt, Germany). We also wish to thank the staffs of the blood bank and the laboratories of the Department of Anesthesiology of Giessen University Hospital for their cooperation.

Received November 19, 2002; accepted February 12, 2003.

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[3] Nonstandard abbreviations: PRBC, packed red blood cell; FHb, free hemoglobin; LDH, lactate dehydrogenase; HBDH, [alpha]-hydroxybutyrate dehydrogenase; FSC, forward side scatter; and SSC, sideward side scatter.

Jan Hirsch, [1] * Axel Menzebach, [1] Ingeborg Dorothea Welters, [1] Gerald Volker Dietrich, [1] Norbert Katz, [2] and Gunter Hempelmann [1]

[1] Department of Anesthesiology and Intensive Care Medicine and [2] Institute of Clinical Chemistry, University Hospital, 35385 Giessen, Germany.

* Address correspondence to this author at: Department of Anesthesiology and Intensive Care Medicine, Universitatsklinikum Giessen, Rudolf Buchheim Strasse 7, 35385 Giessen, Germany. Fax 49-641-99-199-60; e-mail Jan.Hirsch@ chiru.med.uni-giessen.de.
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Title Annotation:Other Areas of Clinical Chemistry
Author:Hirsch, Jan; Menzebach, Axel; Welters, Ingeborg Dorothea; Dietrich, Gerald Volker; Katz, Norbert; He
Publication:Clinical Chemistry
Date:May 1, 2003
Words:5326
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