ERYTHROCYTE VOLUMES AFTER PERFUSION WITH HOMOLOGOUS BLOOD

ERYTHROCYTE VOLUMES AFTER PERFUSION W I T H HOMOLOGOUS BLOOD Richard A. Theye, M.D.,* and John W. Kirklin, M.D.,** Rochester,

Minn.

I

N a series of studies, Litwak, Gadboys, and associates1"3 have introduced and developed the concept that an "homologous blood syndrome" may be as­ sociated with clinical perfusions in which large volumes of whole blood are used to prime the apparatus. The dynamic aspects of this state apparently include temporary sequestration of significant volumes of blood during the perfusion, with reappearance during the first 24 to 48 hours of the postperfusion period. At each time, the resultant changes in total circulatory blood volume are said to predispose to certain undesirable phenomena, primarily cir­ culatory, during the phase of sequestration and pulmonary during desequestration. It is implied that the latter are from hypervolemia and increased pul­ monary venous pressure. Evidence for the occurrence of these events in patients subjected to wholebody perfusion is based primarily upon comparison of serial measurements of blood volume with the record of volume of blood lost externally and volume of blood given intravenously. Sequestration is assumed to have occurred when the measured blood volume is less than that expected from the previous blood volume and the record of intervening blood loss and gain. This is confirmed and desequestration is said to occur when succeeding measurements suggest an augmentation of blood volume greater than that expected from the clinical blood balance. This is a report of a search in patients for changes in measured erythrocyte and blood volumes which are different from those predicted by blood balance data. The postperfusion period was chosen for study since precise measurement of blood loss during the period of open cardiotomy is difficult. In addition, the equilibration time of injected tagged (Cr 51 ) erythrocytes during this period has been observed along with measurements of cardiac output and venous and ar­ terial blood pressures. MATERIAL AND METHODS

Ten patients were studied (Table I ) . All had a period of whole-body perFrom the Mayo Clinic and Mayo Foundation, Rochester, Minn. This investigation was supported in part by Research Grants No. H-3588 and H-4881 from the National Heart Institute, U. S. Public Health Service. Received for publication Jan. 7, 1963. •Section of Anesthesiology. ••Section of Surgery.

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fusion and, except as specifically pointed out, were managed during this time as previously described.4 Patients were considered appropriate for study only if the cardiovascular anatomy permitted the estimation of cardiac output by dye-dilution methods and if postoperative blood loss was not likely to be ex­ cessive. The apparatus was primed with heparinized whole blood without di­ lution in 4 cases. In the others the perfusate consisted of two parts heparinized blood diluted with one part 5 per cent dextrose in water and concentrated serum albumin (80 ml. per 500 ml. of 5 per cent dextrose solution). In the postoperative period, general management was not different from the usual practice. 5 A record was maintained of total postoperative external blood loss (chest drainage plus samples) and replacement (corrected for dilution by ACD solution). The clinical balance for blood was obtained by subtraction of loss from replacement. Erythrocyte (RBC) balance was obtained by multiplying this value by 0.4. Postoperative blood replacement therapy was based in major part upon right atrial pressure (measured via indwelling catheter) and cardiac output (estimated clinically) and only to a minor degree upon the clinical balance.

TABLE I.

T E N PATIENTS STUDIED AFTER PERFUSION W I T H HOMOLOGOUS BLOOD POSTOPERATIVE LOSS t

CASE

1 2 3 4 5 6 7 8 9

AGE ( YEAR)J SEX

14 16 20 7 7 5 7 6 5

M M M M F F M M M

WEIGHT (KG.)

AREA (M.2)

32 56 71 26 18 16 24 23 21

1.16 1.64 1.84 0.94 0.77 0.68 0.88 0.89 0.78

DEFECT*

A. 8.D.

A. S. D. V. S. D. T. O. F. T. O. F. T. O. F. A. S. D. A. S. A. s. r>. and P. S. V. S. D.

PERFUSATEt (LITERS)

4 3.5 3 1.5 2.5 3

2.5 2.5 2.5

(dilute) (dilute) (dilute) (dilute) (dilute) (dilute)

DRAINAGE (ML.)

190 350 690 190 255

1,030

150 160 100

TOTAL (ML.)

490 700

1,170

560 745

1,330

330 350 300

1.48 F 52 23 4 420 650 •Atrial septal defect (A.S.D.), ventricular septal defect (V.S.D.), tetralogy of Fallot (T.O.F.), aortic stenosis (A.S.), pulmonary stenosis (P.S.). tTotal volume of heparinized whole blood used in initiating and completing the perfusion and in immediate postperfusion transfusion. Dilution of this perfusate with 5 per cent glucose in water and albumin is indicated by dilute in parentheses (dilute). tTotal postoperative loss is a summation of loss by drainage from chest tube (first column) and of all other losses (primarily sampling associated with studies).

10

Hemodynamic observations were made using methods* previously de­ scribed.6' 7 Erythrocyte volumes (CV) were determined using a dose of tagged (Cr 5 1 ), unwashed erythrocytes (cell indicator) prepared by the method of Read 8 from fresh whole blood (Strumia-ACD) obtained from the patient to be studied. Plasma volumes (PV) were based upon a separate injection, at another time, of albumin labeled with I 131 (plasma indicator). With few ex•The indocyanine green was kindly supplied by Hynson, Westcott, & Dunning, Inc., Balti­ more, Maryland.

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RESPONSE TO HOMOLOOOUS BLOOD PERFUSION ( T - 23 years, V.S.D.!

Before RADIAL

ARTERY

I

Perfusion

After Perfusion

I

2.5 mg. tnaocyonine Green into Left Atrium

80 RADIAL ARTERY (mm. Hg)

40 LEFT ATRIUM. I mm. Hg)

Fig. 1.—Hemodynamics before (left panel) and after (right panel) replacement of blood with an equivalent volume derived in part (approximately 50 per cent) from 8 homologous donors (see text for details). The patient was a 23-year-old woman with a ventricular septal defect (Case 10). No change in radial artery and left atrial pressures or left atrial flow is apparent.

ceptions all indicators were injected via an atrial catheter. In each instance, concentration of indicator was determined in replicate on serial samples from the radial artery. Sampling was started at 10 to 15 minutes after injection of indicator and was continued over a variable length of time (see Results). CV was calculated from the final concentration of cell indicator and PV from the extrapolated value of plasma indicator. Dry BDTA was the anticoagulant used in sample storage. Blood volume (BV) was obtained by summing CV and PV. Mean circulatory hematoerit (Hm) value was obtained by dividing CV by BV. Central hematoerit (He) value was determined by centrifuging samples for 1 hour at 4° C. and 2,000 g and has been corrected for trapped plasma (factor 0.96). The general laboratory and calculating procedures are those described by Owen.9 In one patient (Case 10) the acute hemodynamic response to homologous blood was measured before and after 5 minutes of perfusion (2.0 L. per minute per square meter) at 37° C. The pump-oxygenator had been primed with 4 L. of undiluted homologous blood obtained from 8 donors. Venous return was obtained by right atrial cannulation and cardiac action remained effective throughout the period of perfusion. The volume of blood in the apparatus was unchanged by the procedure and the net transfusion to the patient was

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TABLE I I .

OBSERVATIONS IN 2 P A T I E N T S FOLLOWING MEASURED

CHANGE* IN CLINICAL BALANCEt (ML.) CASE

DAY

BLOOD

1

1 (OR) 1 2

0 -170 -80

2

i

J. Thora ic and Cardiovas Surg.

KIRKLIN

ACTUAL

RBC

CV

0 -70 -30

860 770 820

1

(ML PV

1,600 1,720 1,320

)

1

BV

2,460 2,490 2,140

1,920 3,090 1 (OR) 0 0 1,170 2,080 3,300 1 +120 +50 1,220 1,940 3,240 2 +230 +90 1,300 *AI1 changes are based upon the relation of the observed value to that observed on Day 1 tEstimated: total blood given (volume corrected for diluent) minus total blood lost. ^Calculated : (0.96) times cell volume/total volume in centrifuged sample of blood.

believed to be zero. Over the period of observation, nasopharyngeal tempera­ ture and levels of anesthetic agents were unchanged and surgical blood loss and mechanical trauma were minimal. The net effect of the procedure was regarded to be replacement, volume for volume, of the patient's blood with a mixture derived in part (approximately one half) from 8 homologous donors. RESULTS

The hemodynamics observed in Case 10 before and after 5 minutes of perfusion are illustrated in Fig. 1. No change was observed in any of the pressures or flows measured. Table II lists the observations in 2 patients in the operating room, 2 hours postoperatively (Day 1), and the next morning (Day 2). Volume measure­ ments are based upon 10 and 15 minute samples. Concentration of tagged erythrocytes at 15 minutes was the same (± 1 per cent) as that at 10 minutes in four of the six observations and less by 2 and 3 per cent in the other two. Excellent agreement between change in clinical RBC balance and measured CV was found. Change in clinical blood balance and BV agree less well but the observed discrepancies are within the possible error of observation. The observations contain no evidence for disappearance or reappearance of erythro­ cytes or sizable volumes of whole blood during the first 24 hours after perfusion. Hm was always lower than He (a normal relationship) and the ratio Hm/Hc was variable. Systemic blood flow was excellent. Between Day 1 and Day 2, changes in right atrial pressure were directionally opposite to those in blood volume. Observations in 8 additional patients during the first 3 postoperative days are listed in Table III. In each instance, change in the clinical RBC balance was associated with a change in measured CV in the same direction (Fig. 2). Unreplaced loss was associated with reductions in measured CV. Replacement in excess of measured loss was associated with an increase in CV. Quantita­ tive agreement between the clinical estimate and the measured change is believed to be within the combined error of the two values. Agreement was similar fol­ lowing the use of a diluted (Cases 3 to 6) or a nondiluted (Cases 7 to 10) perfusate. In Case 6 on Day 3, change in CV from Day 1 exceeded the clinical estimate by 100 ml. This, the largest discrepancy of the series in that direction,

ERYTHROCYTE

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PERFUSION W I T H HOMOLOGOUS BLOOD VOLUMES CHANGE* (ML.) CV

0 -90 -40 0 +50

|

PV

|

0

+120 -280

Het

Hm

0 +30

.41 .41 .42

.35 .31 .38

0

.43 .41 .47

.38 .37 .40

-320

0

+210 +150

+160

+20

+130

BV

Hm/Hc

PRESSURES (MM. H g )

Q L./MIN./M.2

RAD. ART.

.85 .76 .90

3.8 5.5 3.8

109/48 132/62 128/70

7 1 8

.88 .90 .85

3.7 4.2 3.7

130/81 124/64 114/66

12 3 4

| RT. ATRIUM

in the operating room.

was associated with the largest volume of blood loss and replacement. Several observations suggest that these circumstances are conducive to somewhat inaccurate replacement of erythroeytes. The concentration of hemo­ globin in undiluted chest drainage from these patients was always less than that in the peripheral blood. Actual values vary between 5 and 10 Gm. hemoTABLE I I I .

CLINICAL BLOOD BALANCE, MEASURED BLOOD VOLUMES, AND HEMODYNAMICS IN 8 PATIENTS AFTER PERFUSION W I T H HOMOLOGOUS BLOOD CHANGE* IN CLINICAL BALANCEt (ML.) BLOOD

RBC

MEASURED RBC VOLUME (OV) (ML.)

ESTIMATED BLOOD VOLUME § (ML.)

ACTUAL OHANGE*| Ilct

ACTUAL 'CHANGE*

Q MIN./ M.2)

PRESSURES (MM. Hg-) RAD. ART.

L. ATRIUM

3

1 2 3

0 +150 +50

0 +60 +20

1,540 1,560 1,540

0 +20 0

.43 .50 .46

3,920 3,450 3,680

0 -470 -240

3.7 2.7 2.7

153/80 8 131/84 11 120/81 9

4

1 2 3

0 -50 -100

0 -20 -40

640 570 550

0 -70 -90

.44 .46 .41

1,610 1,370 1,500

0 -240 -110

5.5 3.7 5.2

163/89 118/80 142/75

7 6 9

5

1 2 3

0 0 0

0 0 0

410 420 420

0 +10 +10

.45 .48 .45

1,010 960 1,030

0 -50 +20

3.1 1.9 2.6

132/86 95/67 118/81

3 9 6

6

1 2 3

0 +150 +140

0 +60 +60

540 580 700

0 +40 +160

.47 .52 .54

1,250 1,210 1,420

0 -40 +170

3.1 2.5 2.6

106/67 15 (R) 98/69 12 (R) 105/72 15 (R)

7

1 2 3

0 +180 +130

0 +70 +50

660 740 750

0 +80 +90

.42 .49 .45

1,700 1,680 1,850

0 -20 +150

5.6 4.5

119/76 7 125/78 17 97/73 12

8

1 2 3

0 +20 +70

0 +10 +30

530 610 610

0 +80 +80

.42 .41 .40

1,380 1,620 1,690

0 +240 +310

3.5 4.1 4.0

83/53 80/61 81/63

8 15 17

9

1 2 3

0 -50 -20

0 -20 -10

530 500 480

0 -30 -50

.47 .47 .44

1,240 1,180 1,210

0 -60 -30

5.1 3.2

156/74 150/84

5 5

10

1 2 3

0 -210 -290

0 -80 -120

1,130 930 860

0 -200 -270

.46 .46 .38

2,690 2,250 2,500

0 -440 -190

3.3 2.5

136/84 11 116/74 5

•All changes are based upon the relation of the observed value to that observed on Day 1. tEstimated : total blood given (volume corrected for diluent) minus total blood lost. JCalculated: (0.96) times cell volume/total volume in centrifuged sample of blood. SCalculated using measured RBC volume and He (corr.) and assumed Hm/Hc of 0.91.

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globin per 100 ml. and were lowest in patients with minimal drainage. This results in a tendency to overestimate erythrocyte loss. Transfusion practice in these patients includes discarding incompletely emptied units of blood. The discarded mixture usually has a lower concentration of cells than the original mixture. This also results in a tendency to overreplace erythrocytes. Reten­ tion in the thorax of some blood lost from the vascular system and therefore incomplete external recovery would be expected to occur and to result in un­ derestimates of erythrocyte loss. Change in Measured

and E s t i m a t e d

RBC Volume

(Postperfusion)

Clinical balance RBC volume A ml.

Measured(Cr51 RBC volume A m -300

Pig. 2.—Correlation of change in measured (horizontal axis) and clinically estimated (vertical axis) erythrocyte volumes in .the postperfusion period. The differences observed between the first and succeeding daily values in 10 patients are plotted. Complete agreement in direc­ tion of change is present. Quantitative agreement is within the combined error of the two methods. Observations compatible with the concept of reappearance of previously sequestered erythrocytes would be expected to fall to the right of the line of identity and within the shaded area.

Change in estimated blood volume agreed less well with the change in clinical balance (Fig. 3). The observed discrepancies are, however, well within the combined error known to be associated with a clinical blood balance and an estimate of blood volume based upon CV, He, and an assumed factor (0.91 was used) relating Hm and He. Systemic blood flow was not remarkable except in Case 5 on Day 2 when it was low (1.9 L. per minute per square meter). Left atrial pressure was never higher than 17 mm. Hg, and frequently changed directionally opposite to changes in blood volume. In these patients, equilibration time and degree of mixing of injected tagged erythrocytes (indicator), although not listed in the table, were followed (starting at 10 minutes) for at least 30 minutes, usually 45 minutes, and up to 120 minutes after injection. In Case 3 on Day 1, concentration of indicator

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in the nine observations starting at 12 minutes and ending at 120 minutes did not change systematically and all observed values were within ± 1 per cent of the mean. Listed values for CV are based upon the final concentration of indi­ cator observed. Values at earlier times did not deviate by more than 2 per cent in 40, nor by more than 3 per cent in 43 of the 44 observations. The ex­ ceptional deviation (7 per cent) was observed in the 10 minute sample in Case 5 on Day 2 and was in association with the lowest cardiac index (1.9) observed in the study. CV calculated from the concentration of indicator at 10 minutes in this patient would have been 7 per cent lower than that calculated from the concentration observed at 30 minutes. Change

in E s t i m a t e d

Blood

Volumes

Clinical blood

(Postperfusion)

balance yolume

A ml.

Estimated * blood volume A ml.

-500

Tcv

-300.

-1

I

LHcX.9lJ

Fig. 3.—Correlation between change in estimated blood volume (horizontal axis) and clinical blood balance (vertical axis) in the postpeifusion period. The differences observed between the first and succeeding daily values in 10 patients are plotted. The agreement between change in estimated blood volume and clinical blood balance is poorer than that observed for erythrocytes (Pig. 2), but it is well within the combined error of the two methods. Observa­ tions compatible with the concept of reappearance of previously sequestered blood would be expected to fall to the right of the line of identity and within the shaded area. DISCUSSION

Left atrial and arterial pressures and cardiac output did not change follow­ ing acute replacement of blood with an equivalent volume approximately 50 per cent of which was homologous blood. In the first 48 postoperative hours, meas­ ured cell and plasma volumes changed in direction and magnitude as predicted from the record of blood lost and given. Deviations from the predicted were small and within the errors of the methods employed. Even estimated blood volumes, an admittedly unreliable approach to measurement of total blood volume, pro­ vided results which agreed fairly well with the clinical estimates of loss and replacement. Significant sequestration and desequestration is, accordingly, not believed to have occurred in these patients over the period of observation. No support was obtained for the hypothesis that hypervolemia with consequent

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elevation of pulmonary venous pressure is likely to be present 48 hours postoperatively. Litwak and associates1"3 arrived at a different conclusion after a series of experimental and clinical studies. The clinical studies included preoperative and postoperative determinations of blood volume, venous pressure, and body weight, along with a clinical blood balance. It is not clear if single or double indicator techniques were used. Sampling times and tests for thorough mixing were not defined. The hcmodynamic state of the patient was not described. The volumes of blood lost and replaced were not recorded. Failure of agree­ ment between the changes in clinical blood balance and measured blood volume was a major finding and point of argument in those studies. The discrepancy between those studies and the present one is thus, primarily, one of a difference in observations and not of a difference in interpretation. The basis for this discrepancy is not evident at this time. While several possible explanations were suggested by the present study, none was confirmed. In the present study, agreement between change in predicted and measured blood volumes was best when only erythrocyte volume was considered and when blood loss and replacement were minimal and nearly equal. Accuracy of CV determinations was enhanced by serial sampling over enough time to allow complete mixing. In one instance it appeared that a low systemic flow pro­ longed the mixing time significantly. These correlations are to be expected from existing knowledge related to the measurement of blood volume.10 Briefly, the commonly used plasma indica­ tors are distributed in a space without precise anatomic boundaries. In time, all become distributed throughout the extracellular space—both intravascular and extravascular. Since plasma indicator is assvimed to be lost continuously from the intravascular space, the initial distribution used in the calculation of plasma volume requires extrapolation of observed values. Despite extrap­ olation to an assumed purely intravascular space, there is some evidence that this is larger than can be accounted for anatomically. Erythrocyte indicators apparently remain within the intravascular space. Equilibration can be as­ sumed to have occurred when constant concentrations are observed. Extrap­ olation is not required. Certain physiologic states, notably shock, are capable of delaying mixing or even limiting the accessibility to intravascular erythrocytes. 11 Accordingly, in the use of this indicator in the presence of reduced blood flow, hypotension, shock, and so forth, sampling must be carried out over sufficient time to get the final, equilibrated value. Mixing and distribution of plasma indicators is apparently not significantly delayed in these states. SUMMARY

Observations of changes in blood volume and of hemodynamics have been made over the first 3 postoperative days in 12 patients managed with the aid of whole-body perfusion. In a special study in one patient, pressures and flows were not changed with replacement of blood by an equivalent volume derived in part (approximately one half) from 8 homologous donors. The replacement

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was effected by 5 minutes of perfusion without change in the volume of blood in the apparatus. In all 12 patients, good agreement between predicted and measured changes in erythrocyte volumes was obtained. The agreement between predicted change in blood volume and change in estimated blood volume was poorer but within the combined errors of the methods used. No difference was noted between observations following the use of a diluted or a nondiluted perfusate. It is concluded that significant vohimes of erythrocytes or whole blood were not "sequestered" or "desequestered" during the post-perfusion period in these patients. Equilibration time of injected erythrocytes was delayed significantly in the one instance of abnormally low systemic flow rate (1.9 L. per minute per square meter). The relationship of mean circulatory hematocrit value to pe­ ripheral hematocrit value was inconstant and different from that in normal man in the 2 patients studied. It would appear that great care must be exercised in the determination and interpretation of blood volume data in these clinical situations. Tin; technical assistance of Ernest Boyer, James Milde, and Henrietta Cranston in carrying out these studies, and the guidance and material support provided by Dr. W. N. Tauxe of the Section of Clinical Pathology are gratefully acknowledged. REFERENCES

1. Litwak, R. S., Gilson, A. J., Slonim, R., McCune, C. C , Kiem, I., and Gadboys, H. L.: Alterations in Blood Volume During " Normovolemic" Total Body Perfusion, J . THORACIC SURG. 42: 477,

1961.

2. Litwak, R. S., Slonim, R., Wisoff, B. G., and Gadboys, H. L.: Homologous Blood Syn­ drome During Extracorporeal Circulation in Man: The Phenomena of Sequestration and Desequestration, Circulation 26: 752, 1962. (Abst.) 3. Gadboys, H. L., Slonim, R., and Litwak, R. S.: Homologous Blood Syndrome. I. Pre­ liminary Observations on Its Relationship to Clinical Cardiopulmonary Bypass, Ann. Surg. 156: 793, 1962. 4. Theye, R. A., Moffitt, E. A., and Kirklin, J . W.: Anesthetic Management During Open lntracardiac Surgery, Anesthesiology 23: 823, 1962. 5. Lyons, \V. S., DuShane, J. W., and Kirklin, J . W.: Postoperative Care After WholeBody Perfusion and Open lntracardiac Operations: Use of Mayo-Gibbon PumpOxygenator and Browns-Emmons Heat Exchanger, J . A. M. A. 173: 625, 1960. 6. Render, K., Kirklin, J . W., MacCarty, C. S., and Theye, R. A.: Physiologic Studies Fol­ lowing Profound Hypothermia and Circulatory Arrest for Treatment of Intracranial Aneurysm, Ann. Surg. 156: 882, 1962. 7. Rehder, K., Kirklin, J . W., and Theye, R. A.: Physiologic Studies Following Surgical Correction of Atrial Septal Defect and Similar Lesions, Circulation 26: 1302, 1962. 8. Read, R. C.: Studies of Red-Cell Volume and Turnover Using Radiochromium: De­ scription of a New ' ' Closed'' Method of Red-Cell-Volume Measurement, Now Eng­ land J . Med. 250: 1021, 1954. 9. Owen, C. A., J r . : Diagnostic Radioisotopes, Springfield, 111., 1959, Charles C Thomas, Publisher, pp. 152-174. 10. Lawson, H. C.: The Volume of Blood—A Critical Examination of Methods for Its Measurement in Handbook of Physiology, edited by W. F . Hamilton and Philip Dow, Washington, I). C , 1962, American Physiological Society, vol. 1, pp. 23-49. 11. Shoemaker, W. C , and Iida, F . : Studies on the Equilibration of Labeled Red Cells and T-1824 in Hemorrhagic Shock, Surg. Gynec. & Obst. 114: 539, 1962.