Chapter 9

Studies of Blood Volume and Transfusion Therapy in the Korean Battle Casualty*

Captain Theodore C. Prentice, MC, USAR
First Lieutenant John M. Olney, Jr., MC, USAR
Major Curtis P. Artz, MC, USA
Captain John M. Howard, MC, USAR

During the years 1952-53 in the Korean War, there was a trend toward giving increasingly large amounts of blood throughout resuscitation. It was not unusual to administer to the critically injured soldier 15 to 30 pints of blood on the day of injury. Much of the blood was given after the control of obvious hemorrhage. The desirability of this practice was often questioned, but was based on the belief that adequate resuscitation (i. e., stabilization of blood pressure and pulse rate at relatively normal levels, subsidence of clinical symptoms and signs of shock) was principally a function of restoration of blood volume. Likewise, during and after surgery, maintenance of blood volume seemed to be the most critical factor in the recovery or death of the wounded patient. The present studies of blood volume in battle casualties were therefore undertaken in an effort to evaluate these clinical impressions by more objective, quantitative methods, particularly with reference to the desirability and necessity for massive transfusions. All blood used in the Korean Theater was either type O, banked blood or, rarely, fresh compatible blood.

Several previous studies relative to blood volume following wounding have been carried out.^{1, 7, 10} In general, these investigations have stressed primarily the clinical status of the patient as correlated with his blood volume at the time he entered the hospital. For several reasons, we decided to place our emphasis principally on blood volume in the postoperative rather than the early resuscitative phase of the patient's course. (1) It was felt that more could be learned about the adequacy of transfusion and its effect in maintaining blood volume through resuscitation and surgery. In particular, determinations at this time would provide quantitative answers as to the 

*Previously published in Surgery, Gynecology & Obstetrics 99: 542, 1954.

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necessity for large transfusions. (2) In most instances adequate hemostasis was achieved at this time allowing for adequate mixing of the labeled cells or dye without significant loss during mixing. (3) Likewise, the rapid administration of blood or colloids was not necessary here, so that mixing phenomena of labeled cells and dye could be observed without being confounded by concomitant mixing of other rapidly infusing fluids used for resuscitation.

Those patients, therefore, whose blood volumes were determined with labeled red cells, were studied during the first 12 to 48 hours after surgery. Where the dye T-1824 was used it was often necessary to wait until the day following surgery to avoid interfering effects due to elevated plasma hemoglobin.

Methods

Labeled Red Cell Method. Labeling of red cells was carried out using radioactive chromium as the tagging material. Chromium⁵¹ was used in preference to P³² because of the more lasting incorporation of Cr⁵¹ in the red cells as compared to P³². Since with the method used negligible escape of Cr⁵¹ from the red cells occurs within the first 24 hours, prolonged study of mixing could be carried out and serial volumes determined without the necessity of relabeling new cells. The following is the method used for labeling the red cells and calculating the blood volume.

(1) Preparation of labeled red blood cells.

(a) Place 10 ml. of sterile, physiologic saline and 150 to 200 microcuries of radioactive chromium in a sterile, glass-stoppered flask.

(b) Add 15 ml. of fresh heparinized "O" blood to the centrifuge flask.*

(c) Place the flask in an incubator at 38° C. and allow to react for 1 hour, mixing gently every 10 minutes.

(d) Wash the red cells three times with sterile saline.

(e) Suspend the washed red cells in 2 volumes of saline and store at refrigerator temperature until ready for use.

(f) If the cells are to be stored for several hours before use, resuspend in 2 volumes of plasma obtained from bank blood.

(2) Red blood cell volume determination.

(a) Mix the labeled red cell suspension thoroughly and aspirate into a syringe of suitable volume.

*Fresh labeled "O" cells were used in preference to the patient's cells so that a source of labeled cells would be readily available on short notice whenever needed.

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(b) Place at least 2.5 ml. of labeled blood from the syringe into a tared volumetric flask as a standard and weigh. Make to volume with distilled water.

(c) Weigh the syringe containing the material to be injected together with the needle to be used for injection.

(d) Inject the remaining labeled blood intraveneously and weigh the syringe and needle immediately without rinsing.

(e) Collect blood samples 20 and 40 minutes after injection and as indicated thereafter until mixing is complete. In most cases, multiple samples were taken until the last two aliquots differed by less than 5 per cent.

(f) Carefully determine the hematocrit of each sample. At least three hematocrits were obtained on each patient.

(g) Pipette exactly 5 ml. of each whole blood sample into a counting test tube. Likewise, pipette duplicate 5 ml. samples of standard into counting tubes.

(h) Count all standards and samples in a well-type scintillation counter.*

(3) Computations.

The blood volume is calculated using the standard dilution formula C₁V₁ = C₂V₂ or V₂= C₁V₁/C₂where

C₁ = cpm/ml. of injected material
V_l = volume of injected material
C₂ = cpm/ml. of patient's blood
V₂ = volume of patient's blood.

In this instance:

C₁ = cpm/ml. of diluted standard x volume to which standard was diluted
Weight of labeled blood used as standard
V₁ = Weight of syringe and needle before injections minus weight after injection.
C₂ = cpm/ml. of patient's blood after adequate time for mixing of labeled cells has been allowed.
V₂ = Resultant calculated blood volume.
TRCV = Hematocrit x Total Blood Volume.
Plasma Volume = Total Blood Volume minus TRCV.

*Standard scintillation counting technics were used.

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Dye Method. When the dye T-1824 was used for plasma and blood volume determination, the technic of Gregerson et al.^{8, 9} was utilized wherein three plasma samples were taken at 13-15, 30 and 45-60 minute intervals. The dye concentration of each sample was measured and plotted against time. The curve so obtained was extrapolated linearly back to zero time to correct for dye loss during the mixing period. This value at zero time gave the theoretical concentration of dye that would have occurred in the plasma if uniform mixing had been effected at the instant of injection and none of the dye had been excreted. Where abnormal plasma hemoglobin levels or an interfering cloudiness were suspected the samples were discarded. Each reported determination was made on the basis of three valid samples with the exception of those so indicated where extrapolation was on the basis of only two. When only one plasma level was obtained or when a linear plot did not result, the observation was discarded. The total blood volume was calculated from the plasma volume and the hematocrit value. Where simultaneous blood volumes were determined using labeled cells and dye, the two were administered separately via different veins to avoid error through loss during changing of syringes.

In calculating the total blood volume from the plasma volume or red cell volume, no correction factors were used to offset the possible error introduced by differences between venous and total body hematocrit. The estimated normal blood volume used for the dye method was 8 per cent of body weight, and for the labeled red cell method 70 ml./kilo body weight.²

Controls

(1) Radio chromium, red cell tag.

(a) Fresh red cells labeled with Cr⁵¹ and resuspended in 2 volumes of saline showed a transfer of 1.5 per cent of the total radioactivity to the suspending medium in 36 hours. When bank blood plasma was used for resuspension less than 0.5 per cent of the total radioactivity appeared in the supernatant fluids.

(b) Counting of multiple samples of standard Cr⁵¹ solution, a total of 10,000 counts for each sample, gave a standard deviation of 1.6 per cent.

(c) The red cell volumes of several normal individuals were determined as indicated in Table 6. Three duplicate determinations done several days apart gave volumes differing by 0.5, 1.7 and 4.2 per cent of the total. In each instance the second determination was made with cells that had been stored 6 hours after labeling with Cr⁵¹.

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(2) Evans Blue Dye.

Nine patients who received no transfusions had multiple plasma volume determinations during their postoperative course. Using these volumes and the hematocrit to compute the red blood cell volume it was found that the results were constant within an estimated variation of S. D. = ± 5 per cent.

Results

The results are tabulated in Tables 1 through 3.

Careful evaluation of mixing was carried out during the first 60 to 90 minutes in all patients to make sure that the volume was calculated from completely mixed samples. Samples were taken until successive counts differed by less than 5 per cent. Furthermore, because it was suspected that relatively sequestered areas of blood might exist in some of these patients,^{5, 13, 15} later samples were taken in nine individuals. It was reasoned that if such areas existed and they were not completely sequestered, late samples might have mixed into larger volumes which were not apparent in earlier samples. The results of these studies are seen in Table 2. It can be seen that even though the early samples had reached a plateau and agreed with one another within less than 5 per cent, later samples taken at varying intervals revealed slightly greater apparent volumes in most instances. In all but Case No. 1, the later samples also fell within less than 5 per cent of one another.

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Table 1A. Blood Volume Determined with Radioactive Chromium

*In shock.

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Table 1A. Blood Volume Determined with Radioactive Chromium-Continued

*In shock.

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Table 1A. Blood Volume Determined with Radioactive Chromium-Continued

*In shock.

Note.
1. The preoperative volumes of Case No. 6 are not included in the mean for the group.
2. Only the initial postoperative volume of Case No. 7 is included in the mean for the group.

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Table 1B. Blood Volume Determined with T-1824

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Table 1B. Blood Volume Determined with T-1824-Continued

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Table 1B. Blood Volume Determined with T-1824-Continued

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Table 2. Blood Volume as Related to Time of Sampling

The interpretation of these findings is open to some question owing to the possibility of selective destruction of labeled cells. Two patients were given hexamethonium after the early mixing of labeled cells was complete. It was felt that if slowly mixing, stagnant areas of blood existed, the opening of arterioles and increased flow caused by hexamethonium might improve the circulation through such sites with more complete mixing of labeled cells therein. This process, if it occurred, would increase the measured blood volume. In the two instances, sufficient drug was administered to lower the systolic pressure from the range of 120 to 130 down to 90 to 100. Neither individual showed any increase in volume at intervals of 30 to 60 minutes thereafter.

The postoperative blood volume measurements revealed one outstanding result: namely, that large transfusions were a definite necessity in many of these patients and very rarely resulted in over-transfusion. In fact, regardless of the amount of blood received, the vast majority of patients emerged from surgery with some deficit of total blood volume. Of the 25 patients studied with dye none revealed an initial postoperative blood volume greater than normal. In only 3 patients, out of 28 studied with Cr⁵¹ labeled red cells, was over-transfusion present, and in none of these was there any evidence of cardio-respiratory difficulty. This was probably due to the fact that the degree of over-transfusion was minimal. Two of the three over-transfused patients were followed with successive blood volume measurements. In one instance, the red cell volume returned to normal in 9 days and in the other in 7 days. The latter's course, however, was complicated by jaundice and purpura apparently due

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to thrombocytopenia. Of the three over-transfused patients, one had an abdominal wound and the other two had thoraco-abdominal wounds. They received 7,000, 7,000 and 1,500 ml. of blood, respectively.

Clinical State

In general, varying degrees of hypovolemia were tolerated very well by the group during this postoperative period. Fourteen or 58 per cent of the weighed patients studied with labeled cells showed deficiencies of 15 per cent or more of the average normal for their weight. We would class this group as definitely under-transfused. The average deficiency for these patients was 32 per cent. However, of the 14 patients only 3 were in shock, these patients showing deficiencies of 38, 43 and 52 per cent, respectively. The others were doing well postoperatively with no clinical evidence of shock. The average amount of blood which had been received prior to the blood volume determination in this hypovolemic group was 6,740 ml. This figure is in contrast to the group whose deficit was less than 15 per cent. Their average replacement was 3,187 ml. or less than half that of the hypovolemic patients.

The results were even more striking in the patients studied with dye. Fourteen of the eighteen patients whose weights were known and whose normal blood volume could therefore be calculated revealed a deficit of greater than 15 per cent. Their average deficit was 31 per cent. All these patients were doing well postoperatively with no clinical evidence of shock. Their blood requirement pattern was similar to those studied with labeled cells, the group with greater than 15 per cent deficit having received 7,785 ml. and the group with less than 15 per cent deficit receiving 4,160 ml. Thus those individuals who had required the most blood remained the most hypovolemic following surgery. It was in this group that the most massive transfusions were required.

In only one patient did shock exist in the presence of a normal blood volume. This patient had received severe head injuries, with shell and bone fragments in the brain substance. In addition, there were severe facial injuries and a compound comminuted fracture of the left humerus. This patient had received 4,500 ml. of blood prior to the blood volume determination, which measured 96 per cent of normal for his weight. The volume was done 3 hours before his death at which time his pulse was 140-160, respiration 40-50, B.P. 120/100 dropping to 90/70 during the succeeding hour. He died in severe pulmonary edema. The severe brain injury was undoubtedly

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of paramount importance here and may well explain the shock picture in the presence of a normal blood volume.

When the patients are placed in categories dependent upon the location of their wounds, several trends relative to blood volume and blood requirements come to light. In view of the relatively small numbers of patients, these trends must be considered tentative. They seem to be dependent primarily on one fundamental factor, the presence or absence of large areas of muscle injury. In the extremity wounds where a large amount of muscle injury was almost invariably present, large amounts of blood were necessary during resuscitation and surgery. Yet when the blood volume was determined postoperatively by either method, all members of the group fell in the hypovolemic class with deficits in volume of 15 per cent or more and an average hematocrit of 36.5. On the other hand, those who had abdominal wounds, though they had received a similar amount of blood compared to those with extremity wounds, revealed postoperative blood volumes more closely approximating normal and an average hematocrit of 45.8. The excess loss of blood from wound of muscle is easily understood when one considers the local pathology involved in wounds caused by implements of war. Although there may be only a small wound of entrance, there is a large amount of destruction of the underlying muscle. This is particularly true in high-velocity missile wounds. Bleeding from the large mass of damaged muscle continues from the time of injury until operation. During this time, the blood loss is greater than the observer generally realizes. The importance of other factors, such as hemolysis and trapping of blood, as added mechanisms for the causation of these low blood volumes is not fully known at the present time.

Relation of Hematocrit

Table 3 illustrates 10 patients with extremity wounds on whom serial postoperative hematocrits were done. They show a consistent fall in hematocrit. One factor which may contribute in part to this effect is the rise in plasma volume observed in many hypovolemic convalescent patients. A rising plasma volume and falling hematocrit was noted in the absence of any significant change in the total red cell volume indicating a dilution effect as being at least partially responsible for the falling hematocrit.

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Table 3. Hematocrit Changes During Resuscitation, Operation and Convalescence

*Intravenous therapy.

As has been previously reported, hemoconcentration characteristically follows severe intra-abdominal injuries.¹ This is probably due to the loss of plasma in excess of red cells into the bowel wall, mesentery and peritoneal cavity. Serial postoperative hematocrits in patients with abdominal injuries as compared with the postoperative changes following wounds of the extremities, are seen in Table 3. Because hemoconcentration and plasma loss were so frequently remarked, surgeons often administered dextran as an adjunct to blood trans-

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fusion therapy during and after repair of major intra-abdominal injuries. This was true during much of the current observation.

Comparative Blood Volumes

In 15 patients, simultaneous blood volume determinations were carried out using labeled red cells and T-1824. The results are recorded in Table 4. In general the methods agreed fairly well, the average difference being 16.3 per cent and falling in the range of difference found by previous investigators in normal individuals. In three patients (20 per cent), however, the discrepancy was considerably larger (26 to 39 per cent), the dye volume being larger in all instances. All three of these individuals had severe abdominal wounds, two of the three having lacerations involving the liver. This would suggest that the same factor causing hemoconcentration in such patients allows for leakage of dye out of the vascular system with resultant falsely high plasma and blood volume determinations. Peters¹² has commented previously on the loss of the dye, T-1824, from the blood stream particularly in the liver vasculature. The involvement of the liver in two of these three cases is therefore of added interest and significance. These data suggest that under certain circumstances the dye method may not be a reliable one.

Table 4. Comparison of Simultaneous Blood Volumes Determined With Chromium-Labeled Red Cells and T-1824

*This patient tested preoperatively, therefore not included in Table 1A.

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Discussion

The impression was originally gained by those in the North African-Mediterranean Theater¹ that blood loss rather than any other factor was responsible for shock in the wounded patient. Their findings indicated that the presence or absence of shock as well as its degree upon admission to the hospital was directly correlated with the deficiency in blood volume present at that time. The present studies lend support to these findings and extend the concept to the surgical and postoperative period where it is seen that tremendous amounts of blood are often necessary to maintain the blood volume close to normal range. Of the 52 patients studied, 31 or 59.6 per cent required over 10 pints of blood. In other words, over half of the patients studied required complete replacement of their blood volume. Of these 31 patients, 9 required 20 pints or roughly twice their blood volume. This group emphasized well the real necessity in some patients for massive transfusions. The average deficiency of blood volume in these 31 individuals after receiving such massive transfusion was still 25.2 per cent.

In addition to the problem of how large a circulating blood volume was necessary for these patients, was that of how effectively the blood transfused had increased that volume. Possibly present in almost every patient observed but becoming more apparent with the larger volumes of transfusion, was an apparent discrepancy between the amount of blood transfused and that actually measured after operation. This volume deficit appears to consist of both plasma and red blood cells. As examples the data of Table 5 were extracted from Table 1 and arbitrary estimates of admission blood volume made. Most of these patients were chosen as examples because the volumes involved were large enough to make errors in estimation or initial volume relatively insignificant. They demonstrate an average deficit of 5,373 ml. with a range from 1,600 to 7,900 ml. Likewise 10 patients requiring postoperative transfusions were studied with one or more determinations of their plasma volume with Evans Blue for up to 7 days after wounding. The results are tabulated in Table 7. When a transfusion intervened, between two determinations the change in circulatory red cells measured was significantly less than that expected in 6 of 12 instances. None of these patients showed clinical reason to suspect red cell loss.

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Table 5. Showing Discrepancy Between Blood Received and Blood Volume Thereafter

¹Estimated as 40% of normal.
²Volume of whole blood received during resuscitation and operation.
³The circulating blood volume measured several hours to 1 day after operation.
⁴Columns (c) and (d) less column (e).
⁵Measured with Evans Blue and hematocrit.
⁶Measured with Cr^5l and hematocrit.
⁷These patients entered with tourniquets in place above traumatic amputations and underwent uneventful amputations with minimal blood loss and had thereafter only the clean amputation wounds.

Table 6. Red Cell Volumes as Determined With Radio-Chromium

¹Hematocrit determined on the specimens taken for Cr⁵¹ determination.
²Red blood cell volume expected on the basis of 30 ml./Kg. body weight.
³Red blood cell volume measured.
⁴ Female.

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Table 7. Red Blood Cell Volume Response to Transfusion, Measured With Evans Blue

¹Red cells received by transfusions between measurements of the plasma volume.
²The change in red cell volume as measured by plasma volume and hematocrit determinations before and after transfusion.
³The difference between columns (a) and (c).
⁴ Column (d) expressed as per cent of the total measured red cell volume.
⁵Column (d) expressed as per cent of the transfused red cell volume.

It was because of these consistently low blood volumes after large transfusion and the discrepancy between the volume of blood infused and that measured thereafter that such concern was shown over the possibility of incomplete mixing of labeled cells in relatively sequestered areas of blood volume. If large amounts of blood were pooled in areas relatively inaccessible to mixing, falsely low total blood volumes would be the result. Although the blood volumes calculated from late samples were slightly greater than those calculated from early ones, in no instance did they result in a blood volume of 5,000 ml. or over. One of the ten patients showed an increase of 1,565 ml. in blood volume as calculated from the late samples. He had previously been resuscitated with only 2,500 ml. of blood. Of the remaining

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patients, seven received 10,500, 10,000, 8,500, 7,500, 7,000, 3,500 and 3,500 ml. respectively, and the blood volume increase when calculated from the late samples as compared with the early samples was 600 ml. or less in every instance. Likewise in the two patients given hexamethonium no increase in blood volume occurred concomitant with the fall in blood pressure. Furthermore, in those patients who had received massive transfusion and survived, none developed clinical or laboratory evidence of over-transfusion during their postoperative course. If such a pooling mechanism as has been postulated existed early, one would expect a reversal of this process during clinical recovery with mobilization of substantial amounts of trapped blood and some resultant evidence of over-transfusion. In no instance did such occur. Although these data are not sufficient for positive conclusions, they do not support the concept that a significant amount of pooling existed in these patients receiving massive transfusions.

Further answers to this question might be obtained by two approaches. (1) Weigh the patient on an accurate scale when he enters the hospital. When resuscitation and surgery are completed, weigh again and determine the blood volume. Where such large volumes of infused blood are involved, one should see significant gains in weight if most of the blood has been retained; little or no change if it is being lost externally as fast as it is being infused. Correlating the postoperative blood volume with change in weight would thus help clarify the question of loss versus pooling. Obviously if most of the bleeding was into the tissues little would be learned by this procedure. (2) Determine the blood volume postoperatively with chromium labeled cells. Determine the hemoglobin to Cr⁵¹ ratio in blood and in tissue biopsies such as muscle. If large amounts of blood are sequestered in the tissue and inaccessible to the labeled cells, the hemoglobin to chromium ratio will be increased considerably in the tissue.

The quality of bank blood used for transfusion was investigated³ and so far as indicated by the indices used (plasma hemoglobin, plasma potassium and osmotic fragility) this blood was comparable to that available in the United States.

Investigations by means of the Ashby count, as reported previously,³ indicate that in a small portion of A, B and AB recipients receiving large amounts of O blood there will be a destruction of the patient's own cells extending over several days but this is relatively infrequent and usually involves volume changes much smaller than those reported here.

The relative importance of hemolysis of recipient and donor cells as a contributing cause for the large requirement of blood replacement in these patients is not fully known at the present time. These

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patients do not show overt clinical manifestations of rapid blood destruction such as chills, fever, jaundice, etc. Moreover, the average plasma Hg done immediately after surgery in 22 patients who had received an average of 12 pints of blood each day was only 18 mg. per 100 ml.³ (normal 5 mg. per 100 ml.). Since the intravenous infusion of 10 to 16 gm. of hemoglobin (equivalent to hemolysis of 60 to 100 ml. of blood) in normal individuals will induce plasma Hg levels of greater than 300 mg. per 100 ml.,^{6, 11} the levels found in these postoperative patients would be indicative of minimal intravascular hemolysis. The rate of plasma hemoglobin clearance is of course a factor here. The most rapid clearance rate found in the group studied was 5 mg. per 100 ml. per hour, which would be insufficient to lower a significantly high concentration of hemoglobin to the levels found even over a period of many hours. Likewise the observed bilirubin levels in a similar group of patients¹⁴ would correspond to a relatively small degree of blood destruction.

The rate of plasma clearance, principally by the liver, is again a critical factor here and the necessary data for such evaluation in these patients are not at hand. The infusion of 16.4 gm. of hemoglobin⁶ (equivalent to hemolysis of approximately 100 ml. blood) in a normal individual resulted in a plasma bilirubin concentration of 1.4 mg. per 100 ml. 10 hours later, with a gradual fall thereafter and remaining greater than the pre-injection level 24 hours later. Clearance of bilirubin in the wounded patient, where impaired hepatic function has been shown by others ^{1, 14} would be expected to be slower than in normals. Therefore the 6-hour postoperative average level of 2.5 mg. per 100 ml. which is the highest level reached in any of the preoperative and postoperative specimens would imply relatively small amounts of hemolysis.

The postoperative hematocrit afforded a relatively poor index to the requirements for transfusion. Because of rapid changes in plasma volume which take place independently from changes in red cell volume after extensive blood loss, the hematocrit gives little information about the actual red cell volume at any given time. By the same token, any conclusions concerning the status of total blood volume drawn from hematocrit data alone are likely to be in error.

Conclusions

Blood volume determinations have been carried out postoperatively in 52 wounded patients. These patients required large amounts of blood to maintain them through the phases of resuscitation and surgery. Generally those with extremity wounds, where large amounts of muscle destruction had taken place, required the largest amounts

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of blood and still remained the most hypovolemic following surgery. However, no matter where the injury was located, if considerable areas of muscle were involved, large amounts of blood were usually required.

Over-transfusion occurred in only three instances and in no case was it of sufficient degree to cause cardio-respiratory symptoms. The error in most cases was under-transfusion rather than over-transfusion.

Hematocrit determinations in the postoperative period are not a reliable index to the requirement for blood.

Where simultaneous blood volumes were determined with labeled cells and dye, the difference between the two was 16 per cent, the dye volume being greater in all but four instances. However, in severe abdominal wounds, especially with liver involvement, the discrepancy was much larger. Here the dye volumes were greater by 26.8, 33 and 39 per cent. This probably represents increased capillary permeability and gross vascular damage in the involved area with resultant leakage of dye.

The discrepancies between the large amounts of blood given and the small blood volumes determined thereafter are explainable on three possible bases: (1) Trapping of large amounts of blood in a sequestered state which mixes slowly or not at all. (2) Hemolysis of large amounts of donor and/or recipient erythrocytes. (3) Continued loss of blood either externally or into the tissues during the preoperative, operative and postoperative periods. The latter is believed, at the present time, to be the most important of these factors.

References

1. Beecher, H. R., Editor: The Physiologic Effects of Wounds, p. 45. U. S. Government Printing Office, Washington, D. C., 1952.

2. Berlin, M. I., Lawrence, J. H., and Cortland, J.: The Blood Volume in Chronic Leukemia as Determined by P³² Labeled Red Blood Cells. J. Lab. and Clin. Med. 36: 435, 1950.

3. Crosby, W. H., and Howard, J. M.: The Hematologic Response to Wounding and to Resuscitation Accomplished by Large Transfusions of Stored Blood. Blood 9: 439, 1954. (Chapter 6, this volume.)

4. Nachman, H., James, C. W., III, Moore, J. W., and Evans, E. I.: A Comparative Study of Red Cell Volumes in Human Subjects with Radioactive Phosphorus Tagged Red Cells and T-1824 Dye. J. Clin. Invest. 29: 258, 1950.

5. Gibson, J. G., 2nd., Seligman, A. M., Peacock, W. C., Fine, J., Aub, J. G., and Evans, R. D.: The Circulating Red Cell and Plasma Volume and the Distribution of Blood in Large and Minute Vessels in Experimental Shock in Dogs, Measured by Radioactive Isotopes of Iron and Iodine. J. Clin. Invest. 26: 126, 1947.

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6. Gilligen, R. D., Altschule, M. D., and Batersky, E. R.: Studies of Hemoglobinemia and Hemoglobinuria Produced in Man by Intravenous Injection of Hemoglobin Solution. J. Clin. Invest. 20: 177, 1941.

7. Grant, R. T., and Reeve, E. B. Observations on the General Effects of Injury in Man, p. 228. Medical Research Council Special Report, Ser. No. 277, H. M. Stationery Off., London, 1951.

8. Gregersen, M. I., Gibson, J. G., and Stead, E. A. Plasma Volume Determinations with Dyes: Errors in Colorimetry; Use of the Blue Dye T-1824. Amer. J. Physiol. 113: 54-55, 1935

9. Gregersen, M. I.: A Practical Method for the Determination of Blood Volume with the Dye, T-1824; Survey of the Present Basis of the Dye-Method and Its Clinical Applications. J. Lab. and Clin. Med. 29: 1266-1286, 1944.

10. Noble, R. P., and Gregersen, M. I.: Blood Volume in Clinical Shock. II. The Extent and Cause of Blood Volume Reduction in Traumatic Hemorrhage and Burn Shock. J. Clin. Invest. 25: 172, 1946.

11. Ottenberg, R., and Fox, C. I., Jr.: The Rate of Removal of Hemoglobin from the Circulation and Its Renal Threshold in Human Beings. Amer. J. Physiol. 123: 516, 1938.

12. Peters, J. P.: Role of Sodium in Production of Edema. New Eng. J. Med. 239: 353, 1948.

13. Root, G. T., and Mann, F. C.: An Experimental Study of Shock with Special Reference to Its Effect on the Capillary Bed. Surgery 12: 861, 1942.

14. Scott, R., Jr., Olney, J. M., Jr., and Howard, J. M.: Hepatic Function of the Battle Casualty. (Chapter 9, Volume I of this series.)

15. Zweifach, B. W., Hershey, S. O., Howenstine, E. A., Loe, R. E., Hyman, C., and Chambers, R.: Omental Circulation in Morphinized Dogs Subjected to Graded Hemorrhage. Ann. Surg. 120: 232-250, 1944.

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