Autotransfused shed mediastinal blood has normal erythrocyte survival

Autotransfused Shed Mediastinal Blood Has Normal Erythrocyte Survival Henrik Schmidt, MD, Jens Otto Lund, MD, and Steen Levin Nielsen, M D Department of Anaesthesiology, Gentofte Hospital and Department of Clinical Physiology and Nuclear Medicine, Gentofte and Herlev Hospitals, University of Copenhagen, Hellerup, Denmark

Background. Autotransfusion of shed mediastinal blood may reduce the need for homologous blood transfusions in cardiac surgery. In an earlier study we have shown that the red blood cells (RBCs) of shed mediastinal blood have a normal membrane stability (osmotic fragility) compared with circulating RBCs after coronary artery bypass grafting and better than stored RBCs. This indicates that RBCs in shed mediastinal blood are not damaged further during salvage. It remains to be determined how autotransfusion affects the survival of RBCs from shed mediastinal blood. Methods. We performed a prospective, randomized, and controlled study involving 26 patients having elective, uncomplicated coronary artery bypass grafting. Dual-isotope labeling technique (chromium 51 and techne-

tium 99m) was used to investigate the 24-hour survival of RBCs from shed mediastinal blood and RBCs from circulating blood, and to estimate the mean survival time of RBCs. Results. There was no significant difference between the 24-hour survival of shed mediastinal RBCs and circulating RBCs. The estimated mean cell lifespan was 20.5 days (range, 11.6 to 29.0 days) for shed mediastinal RBCs and 22.7 days (range, 14.4 to 36.2 days) for circulating RBCs. Conclusions. The survival of RBCs from shed mediastinal blood after autotransfusion is comparable with the survival of RBCs in the patients' circulating blood.

of shed mediastinal blood has been A utotransfusion used to reduce the blood requirement after coro-

Material and M e t h o d s

nary artery bypass grafting (CABG) [1-4]. During cardiopulmonary bypass the red blood cells (RBCs) are damaged, with an increase in plasma hemoglobin at the end of cardiopulmonary bypass [1, 5, 6]. During salvage shed mediastinal blood has been in contact with various tissue types and exposed to suction, which might damage the blood cells. Several trials have shown that the concentration of free hemoglobin is elevated in shed mediastinal blood [5-8]. Activation of the complement system, which occurs during autotransfusion, may also induce RBC damage [5]. However, in an earlier study [9] we have shown that the membrane stability of RBCs (normal osmotic fragility) collected by a hardshell cardiotomy reservoir is comparable with that of circulating RBCs after CABG and better than that of stored RBCs. This indicates that RBCs saved from shed mediastinal blood may not be damaged further during salvage. The survival of shed mediastinal RBCs, however, has not been thoroughly evaluated. The aim of the present study was to determine the survival of RBCs from shed mediastinal blood after autotransfusion compared with the survival of normal circulating RBCs in patients undergoing CABG.

Accepted for publication Feb 23, 1996. Address reprint requests to Dr Schmidt, Departmentof Anaesthesiology, Gentofte Hospital,Niels Andersensvej 65, DK-2900HeUerup,Denmark. © 1996 by The Society of Thoracic Surgeons Published by Elsevier Science Inc

(Ann Thorac Surg 1996;62:105-8)

Twenty-six adult patients undergoing primary and uncomplicated CABG entered the study. Inclusion criteria were primary CABG and age between 18 and 80 years. The study was approved by the regional ethical committee. Information was given to each patient and written consent was obtained according to the guidelines of the regional ethical committee. The number of patients in the study was calculated to allow detection of a 10% difference in 24-hour survival of RBCs between shed mediastinal RBCs and circulating RBCs, with a risk of overlooking a real difference of 5% (type I error) and a risk of 10% of accepting a false difference (type II error). Not included in the study were patients with prolonged bleeding time (the Ivy method > 10 minutes), emergency operation, preoperative left ventricular ejection fraction less than 0.40, diabetes mellitus, and pulmonary or kidney diseases. Excluded from the study were patients needing operation for bleeding within the first 18 postoperative hours and patients receiving homologous blood transfusion intraoperatively or within the first 24 hours. If the patients received homologous blood transfusion later during the study, the data after transfusion were not used in the calculations of the lifespan of RBCs. Anesthesia was induced with fentanyl, midazolam, pancuronium, and enflurane in oxygen. The patients were monitored with radial artery, central venous, and pulmonary artery catheters. All operations were performed through a median sternotomy using standard techniques for cardiopulmonary bypass with a crystalloid prime and a delivery rate of 2.4 L • min -1 • m -2. At the 0003-4975/96/$15.00 PII S0003-4975(96)00219-6

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end of the operation all patients had their mediastinal and~ if necessary, pleural tubes attached to the inlet port of a Baxter Cardiotomy/Auto-transfusion reservoir (Baxter Healthcare Corp, Irvine, CA). Mediastinal shed blood was filtered through a 40-/~m filter in the cardiotomy reservoir before autotransfusion. At arrival in the intensive care unit the patients were randomly allocated to three groups before the start of autotransfusion: Group A (10 patients) had circulating RBCs labeled with technetium 99m (99mTc) and shed mediastinal RBCs labeled with chromium 51 (51Cr). Group B (10 patients) had circulating RBCs labeled with 51Cr and shed mediastinal RBCs labeled with 99mTC. Group C (6 patients) served to compare the behavior of the two radionuclides as RBC markers. Three patients had RBCs from circulating blood labeled with both 99mTC and 51Cr and 3 patients had RBCs from shed mediastinal blood labeled with 99mTc as well as 51Cr. Before the start of autotransfusion a 20-mL sample of arterial blood (circulating blood) and a 20-mL sample from the cardiotomy reservoir (shed mediastinal blood) were collected in heparinized syringes. For 99mTClabeling, 2 mL of blood was incubated in a vial with pyrophosphate and stannochloride (CIS TCK11, CIS Bio International France) for 10 minutes. Sodium hypochlorite 0.1%, 0.2 mL, and EDTA 4.4%, 0.05 mL, were added, followed by 800 MBq of a fresh eluate of 99mTc-pertechnetate. The labeling efficiency was measured, and if it was less than 95% erythrocytes were washed in isotonic saline solution. For 5~Cr labeling, erythrocytes from 10 mL of blood were isolated by centrifugation at 1,500 rpm and washed two times in isotonic saline solution. Five MBq 51Cr-sodium chromate was added and cells were incubated for 30 minutes at 37°C during repeated mixing. After three washings in isotonic saline solution the erythrocytes were ready for injection. The two labeled samples of 99mTc-labeled and 51Crlabeled erythrocytes were injected into the patient and the autotransfusion of shed mediastinal blood was stopped (4 hours after the salvage procedure was started). Five-millillter samples of whole blood were collected in heparinized tubes at 15, 30, 45, and 60 minutes and 3, 6, 18, and 24 hours and daily thereafter for approximately 4 days. Three milliliters of each sampie was lysed by cooling to -20°C and counted in a dual-channel automatic gamma counter (COBRA II Autogamma; Packard) along with standards. The count rate for each radionuclide was corrected for background, overlap of the gamma ray spectra, decay during elapsed time for counting, and RBC volume using standard techniques. We used the 15-minute value as the 100% value (early time values averaged method [10]), and a log regression line was calculated for each patient for 99mTc-labeled as well as 51Cr-labeled erythrocytes. From the regression line the 24-hour survival fraction was calculated. The mean cell life span for the SlCr-labeled RBCs was estimated from the 4-day samples. The data from group C were used to calculate a correction factor for 99r"Tc release from the erythrocytes using the following formula: correction factor = 24-hour survival of 51Cr-labeled erythrocytes/24-hour survival of

Ann Thorac Surg 1996;62:105-8

Table 1. Preoperative and Operative Data of the Three Patient Groups" Variable

Group A (n = 10)

Group B (n = 10)

Group C (n = 6)

60 (39-70) 61 (46-68) 57 (50-64) age (y) Left ventricular 0.65 (0.44-0.88) 0.56(0.43--0.82) 0.64(0.50-0.75) ejection fraction Time on CPB 119 (75--180) 116 (57-144) 99 (92-112) (mtn) Aortic cross-clamp 77 (60-140) 75 (26-97) 69 (61-87) time (rain) Number of grafts 4.5 (3-8) 4.5 (3-6) 4.0 (3-5) Operative blood 460 (200-600) 380 (190-550) 496 (300-650) loss (mL) a Data are shownas the median with ranges irt parentheses. There were no significant differencesbetween the groups. CPB = cardiopulmonarybypass.

99mTc-labeled erythrocytes. A corrected 24-hoUr survival fraction of 99~Tc-labeled erythrocytes in groups A and B was calculated by multiplying the percentage survival at 24 hours by the correction factor. Data are expressed as medians with ranges in parentheses. Statistical analysis was performed using the Mann-Whitney test, Wilcoxon paired test, and Kruskal~ Wallis test where appropriate. A p value less than 0.05 was regarded as significant (two-sided tests). Results There were no differences between the groups regarding demographic characteristics and operative data before entering the study (Table 1). None of the patients were excluded due to reOperation for bleeding or blood transfusion within the first 24 hours. Three patients in group A (1 on postoperative day 3 and 2 on postoperative day 4), 1 patient in group B (postoperative day 4), and none in group C received homologous blood transfusion later during the study period. The labeling efficiency of shed mediastinal RBCs with 99mTc was 95% (90% to 98%) compared with 96% (90% to 97%) for circulating RBCs. This indicates that the 99mTc label is equally bound to shed mediastinal RBCs and circulating RBCs, and is therefore a valid marker for autotransfused RBCs. The 24-hour survival of labeled RBCs in group C (Table 2) shows, however, a low in vivo stabili~ for 99mTclabeled RBCs. The 24-hour correction factor to adjust 99mTc-labeled RBC survival was estimated at 1.55 (1.42 to 1.67). There was no significant difference between groups A and B regarding 24-hour survival of shed mediastinal RBCs and circulating RBCs. When the correction factor was used to correct the 99~Tc values there was no significant difference between RBC survival fraction from the two sources (Table 3). Using the correction factor we found no significant difference between the 24-hour survival of transfused shed mediastinal RBCs (median, 92.2%; range, 63.8% to 99.1%) and circulating RBCs (median, 94.1%; range, 61.7% to 116%) (n = 20; Wilcoxon paired test, p = 0.29).

A n n Thorac Surg 1996;62:105-8

SCHMIDT ET AL A U T O T R A N S F U S I O N A N D SURVIVAL OF RED BLOOD CELLS

Table 2. Twenty-four-Hour Suroival of Red Blood Cells From Shed Mediastinal Blood and From Arterial Circulating Blood in Group C (n = 6)

24-Hour 24-Hour Survivalof Survivalof 99mTc-labeled SlCr-Labeled Correction RBCs (%) RBCs (%) Factor

Type of B l o o d Labeled With Both Markers RBCs from SMB RBCs from SMB RBCs from SMB Circulating RBCs Circulating RBCs Circulating RBCs Median Range

62.3 66.1 53.5 58.1 62.1 56.8 59.8 53.5-66.1

94.4 93.7 89.1 91.4 93.4 92.9 92.9 89.1-94.4

51Cr = c h r o m i u m 51; RBCs = red blood cells; mediastinal blood; 99mTc = technetium 99m.

1.52 1.42 1.67 1.57 1.50 1.64 1.55 1.42-1.67 SMB = shed

The survival of 51Cr-labeled shed mediastinal RBCs and circulating RBCs throughout the study are shown in Figure 1. There were no significant differences between the groups. The estimated mean cell lifespan had a median of 20.5 days and a range of 11.6 to 29.0 days for shed mediastinal RBCs and median of 22.7 days and a range of 14.4 to 36.2 days for circulating RBCs (p = 0.15). Comment

This prospective study shows that autotransfused erythrocytes from shed mediastinal blood have a normal survival fraction compared with the patients" circulating RBCs. The demand for blood products has increased with the increasing frequency of heart operations. Transfusion of autologous blood is one approach to reduce the requirements for homologous blood transfusion. Autotransfusion of shed mediastinal blood was first investigated by Schaff and associates in 1978 [1]. Our group has previously shown, with clearly defined criteria for transfusion and volume therapy, that postoperative autotransfusion of shed mediastinal blood after elective uncomplicated Table 3. Twenty-four-Hour Survival of Transfused Shed Mediastinal Red Blood Cells and Circulating Red Blood Cells in Groups A and B a

Group

Corrected 24-Hour 24-Hour 24-Hour Survival of Survivalof Survivalof ~Tc-labeled ~mTc-labeled SlCr-labeled P RBCs (%) RBCs (%) RBCs (%) Valueb

A

60.9(39.8-7.5.2) 94.4(61.7-116) 91.8(81.6-9712) 0.42 (n = 10)

B

(n = 10) p Value

59.9 (41.2-63.9) 92.9(63.8-99.1) 94.3(90.5-96.1) 0~54 0.94

NT

0.15

a Values are shown as m e d i a n s with r a n g e in parentheses, b Wilcoxon paired test b e t w e e n 24-hour survival ofSlCr-labeled RBCs a n d corrected 24-hour survival of ~ T c - l a b e l e d RBCs. ¢ M a n n - W h i t n e y test bet w e e n groups A a n d B. 51Cr = c h r o m i u m 51; NT = not tested; 99mTc = technetium 99m.

RBCs = red blood cells;

107

% survival

looJ 90 80 70

60 50

20

40

60

80

100

hours ~-Shed Medlastinal RBC 4-Circulating RBC

Fig 1. Survival of chromium 51-labeled red blood cells (RBC) of retransfused shed mediastinal blood (group A and 3 patients from group C, n = 10) and circulating blood (group B and 3 patients from group C, n = 12) during the study (median with range).

primary CABG reduces the number of patients needing homologous blood transfusion from 54% to 28% [5]. It is important to ensure that the quality of shed mediastinal blood is acceptable. Transfused RBCs that survive the first 24 hours are believed to have a normal lifespan distribution [11, 12], and measurement of 24hour survival fraction is a valid indicator of RBC survival after transfusion. The use of a dual-isotope labeling technique enabled us to study the 24-hour survival fraction of two populations of RBCs in the same individual. This technique is well accepted [13,14]. The calculation of 24-hour survival fraction by the early time values averaged method has been evaluated by Marcus and colleagues [10]. They showed that this method was equal with the double-isotope methods and the extrapolation methods. In the study by Marcus and colleagues [10] a correction factor of 1.23 was found compared with 1.55 found in the present study. This difference can be explained by the different source of RBCs for 9 9 m T c labeling. Marcus and colleagues [10] used autologous RBCs stored for 42 to 49 days, whereas we used autologous mediastinal shed blood and circulating autologous blood without storage. Stored RBCs have low values of adenosine triphosphate and are deprived of 2,3diphosphoglycerate whereas shed mediastinal blood has normal values of adenosine triphosphate and 2,3diphosphoglycerate [9, 15]. Heaton [16] showed that posttransfusion recovery of 99mTc/S1Cr-labeled RBCs was positively correlated to red cell adenosine triphosphate level (r = 0.6). This could explain the difference between our findings and the findings of Marcus and colleagues [10]. When we used the correction factor each patient served as his or her own control. We were unable to detect a difference between survival of shed mediastinal RBCs used for autotransfusion and survival of circulating RBCs postoperatively after cardiopulmonary bypass. The long-term survival of SlCr-labeled RBCs from the two

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sources after c a r d i o p u l m o n a r y bypass also showed no significant differences. The survival of autotransfused RBCs harvested by various methods has b e e n studied by others. Four studies [17-20] compared the survival of RBCs collected d u r i n g aortic reconstructive procedures with m a t c h e d controls u s i n g cell-saving techniques a n d 51Cr-labeled RBCs. However, these studies suffered from a n u m b e r of methodologic drawbacks. Most important is that the survival of saved RBCs was not compared with the survival of n o n s a v e d RBCs in the same patient or u n d e r the same conditions. The dual-isotope labeling technique has b e e n used in two other studies. In an elegant study of 20 patients, Ansell a n d associates [21] f o u n d no significant difference b e t w e e n survival of RBCs saved intraoperatively with a cell-saving device a n d blood collected preoperatively by venipuncture. In that study [21] the 24hour survival was not calculated, b u t the long-term survival results are comparable with our findings. In the study b y Kent a n d associates [22] on 6 patients the 24-hour survival percentage of salvaged RBCs was 81% with a range of 48% to 94%, which was not different from that of preoperatively collected RBCs. O u r finding of a 92% (range, 81% to 97%) 24-hour survival of RBCs from shed mediastinal blood suggests that blood collection by the cardiotomy reservoir is less d a m a g i n g than use of the cell-saving technique. The alternative to autotransfusion is transfusion of autologous stored blood. The 24-hour survival of RBCs stored for 4 to 5 weeks is about 80% [10, 16, 23, 24]. Autologous RBCs stored for I to 5 days have a 92% to 94% 24-hour survival [10, 12], which is comparable with the results of our findings. It m a y be considered a limitation of the present study that the RBCs from the cardiotomy reservoir were harvested approximately I hour after the start of salvage a n d before the start of autotransfusion. We did not investigate the survival of RBCs salvaged later d u r i n g the autotransfusion period. However, in a n earlier study we have shown that the m e m b r a n e stability of RBCs (osmotic fragility) from shed mediastinal blood was comparable after 1 a n d 6 hours of autotransfusion [9]. The data from our study show that RBCs saved from shed mediastinal blood have not b e e n d a m a g e d by collection with the cardiotomy reservoir. The survival of RBCs from shed mediastinal blood is comparable with the survival of fresh autologous blood.

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