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Autotransfusions of previously frozen blood in elective gynecologic surgery
Autotransfusions
of previously frozen blood
in elective gynecologic surgery THOMAS C.
A.
ROBERT
ROBERT Chetsea,
K.
DAANE,
COMMANDER
(MC)
VALERI,
COMMANDER
BARTON,
CAPTAIN
(MC) (MC)
USN* USNR USN**
Massachusetts
Autotransfusions of previously froten red cells in patients undergoing elective gynecotogic surgery are reported. When red cell volume and total circulating hemoglobin levels are used as criteria, a 4 week interval between the initial and second bleed proved better than a 2 week interval for the collection of two units of blood. The clinical responses to the autotogous transfusions were satisfactory.
blood is limited by its relatively short shelflife : acid-citrate-dextrose (ACD) collected blood can be satisfactorily stored at 4“ C. for no more than 21 days, while adenine supplementation of the ACD medium (AGDadenine) extends the storage period at 4’ C. up to 35 days. *I 21 Preservation of human red cells by freezing permits storage for prolonged periods and thereby lends itself to less rigidity in the scheduling of the bleeding and the surgery. This study was undertaken in order to evaluate the feasibility of a freeze-preservation approach to providing autologous blood for elective gynecologic surgery. At the same time, an attempt was made to ascertain the optimum bleeding schedule for collection of two units of blood prior to elective surgery. The red cells were preserved with highconcentration glycerol and the slow freezethaw technique. The red cells were washed by the Huggins technique, which entails dilution of the glycerolized red cells with nonelectrolyte solutions and recovery by agglomeration, I41 I5 Previous extensive clinical evaluation of preserved red cells by means of small aliquot autotransfusions to healthy volunteers, multiple (two or three) unit homologous transfusions to stable, anemic recipients, and multiple (ten or more)
THEUSEOFHOMOLOGOUS compatible blood to replace surgical blood loss is attended by two major hazards: isosensitization to red cells, white cells, or platelets, and the transmission of disease, particularly serum hepatitis and malaria.4p 5l 8* lo, 171 I91 28 To circumvent these difficulties, autologous blood transfusion has been frequently recommended for elective surgery.3* 7* g, I19 I81 20 For this purpose, the use of liquid preserved
From the Department of Obstetrics and Gynecology, Naval Hospital, and the Naval Blood Research Laboratory. This work was supported by the U. S. Navy. The opinions or assertions contained herein are those of the authors, and are not to be construed as oficial or reflecting the views of the Navy Department or the Naval Service at large. This paper was presented in part at the Armed Forces District Meeting, American College of Obstetrics and Gynecology, Las Vegas, Nevada, Oct. 23, 1967, and District Z Meeting, American College of Obstetrics and Gynecology, Boston, Massachusetts, Oct. 25, 1967. ‘Present address: Department Obstetrics and Gynecology, of California at Los Angeles, Angeles, California 90024. **Present address: Military Assistance APO San Francisco,
Headquarters, Command, Californta
of University Los U. S. Vietnam, 96222.
394
Volume
105
Number
3
Autotransfusions
Table I. Diagnoses of the patients studied in three groups G7OU
4
No.
Group A-11 patients (two-week bleedings) Mean age, 38.3 yr. Mean parity, 4.6 Pelvic relaxation Myoma of uterus Carcinoma in situ of cervix Ch.ronic pelvic inflammatory disease Group .I-12 patients (four-week bleedings) Mean age, 38.6 yr. Mean parity, 4.5 Pelvic relaxation Myoma of uterus Carcinoma in situ of cervix Control group-10 patients (no Mean age, 37.2 yr. Mean parity 3.8 Pelvic relaxation Myoma of uterus Carcinoma in situ of cervix Chronic pelvic inflammatory disease
interval
between
5 3 2 1 interval
between
8 2 2 bleedings)
5 3 1 1
homologous transfusions to severely wounded servicemen in South Vietnam, showed this blood to be safe and efficacious.2* 2ol22-24 Mat,erials
and
methods
Selection and grouping of patients. All of the patients were candidates for elective gynecologic surgery. Table I details the mean age, mean parity, and diagnoses of the patients in each of three groups. Patients in Groups A and B were bled on two occasions. On each occasion approximately !WO ml. of whole blood was collected in 75 ml. ACD (National Institutes of Health, Formula A). In Group A there was a 2 week interval between the two bleedings, whereas in Group B 4 weeks intervened between bleedings. In both groups the elective surgery was performed 4 weeks after the second bleed. The patients in Group C were not bled, serving as the controls for the evaluation of pre- and postoperative hematocrits in patients in Groups A and B undergoing surgery for similar disorders. All of the patients in Groups A and B received
of frozen
blood
395
a high protein diet and 150 Gm. of ferrous fumarate* (approximately 50 mg. of elemental iron) twice daily following the first bleeding. The patients with pelvic relaxation had vaginal hysterectomy and/or culpoplasty and/or culpoperineoplasty; those with leiomyomata uteri, carcinoma in situ of the cervix, and chronic pelvic inflammatory disease had abdominal hysterectomy with or without removal of the adnexa. Red cell volume measurements. Chromium-51 red cell volumes were measured in each patient in Groups A and B immediately prior to the first and second bleedings, and on the day before the elective operation. At the time of the first measurement, 20 ml. of whole blood was collected in 3.0 ml. of modified ACD medium.t Five microcuries (PC) of Naz5*Cr0,$ was added and the blood was incubated at 37O C. for 30 minutes.12 Ascorbic acid was not added nor were the labeled erythrocytes washed.25 A 10 ml. aliquot of the labeled red cells was injected into the patient and a sample collected 30 minutes later. The red cell volume was then reckoned from the observed dilution of the injected red cells’ radioactivity. The red cell volume was measured the second time with 12 to 15 PC of 51Cr, and the third time with 25 ,.LCof 51Cr. A correction for radioactivity in the recipient red cells was required for the second and third red cell volume measurements. The mean corpuscular hemoglobin concentration (MCHC), measured in grams per cent, was derived from the microhematocrit and the total hemoglobin concentration, measured on the day of the red cell volume determination (in milliliters) .61I6 The total cellular hemoglobin level was calculated by multiplying the red cell volume by the MCHC. Red cell preservation. The ACD-collected blood was stored at 4O C. for up to five days. Each unit was then centrifuged in a PR-2 refrigerated centrifuges at 3,500 f 200 *Ferrosequels,
Lederle
Laboratories.
iSquibb ACD solution mod&d (IO ml.) containing citric acid 80 mg., sodium citrate 250 mg., and dextrose anhydrous 120 mg. &Squibb
Chromotope.
$International sachusetts.
Equipment
Company,
Needham,
Mas-
396
Daane,
Valeri,
and Barton
October 1, 1969 Am. J, Obst. &Gym.
r.p.m. for six minutes, following which the plasma was removed and stored at -20° C. The packed red cell mass was transferred to a Blood Freezing Unit* and glycerolized in a Huggins Cytoglomerator* with an equal volume of 8.6M glycerol containing 0.3 per cent disodium ethylenediaminetetra-acetic acid (Na, EDTA), 8 per cent glucose, and 1 per cent fructose. The glycerolized red cell mass was slowly frozen, and stored at -80’ C. for up to two months. On the day of the elective operation each unit was thawed in the 40’ C. water bath of the Huggins Cytoglomerator for approximately five minutes. Each thawed, glycerolized red cell mass was diluted during continuous agitation, first with 500 ml. of 50 per cent dextrose in water, and then with approximately 2,000 ml. of a nonelectrolyte solution containing 8 per cent glucose and 1 per cent fructose. When agitation was stopped the red cells sedimented by spontaand the supernatant neous agglomeration, was expressed into the appropriate section of the Blood Freezing Unit. The red cells were washed twice more with two additional 2,000 ml. volumes of the nonelectrolyte solution, each time being recovered by spontaneous agglomeration when the agitation ceased. The agglomerated red cell mass was disaggregated with 250 ml. of isotonic saline solution. The disaggregated red cells were placed in the PR-2 refrigerated centrifuge and spun at 3,500 t 200 r.p.m. for six minutes, after which the supernatant fluid was removed completely and the thawed autologous plasma added. Approximately 50 minutes was required for the preparation of the two units of blood. The final hematocrit ranged from 25 to 49 with a mean of 32 per cent and a standard deviation of _+ 4.7. The previously frozen red cell mass resuspended in autologous plasma was then stored at 4O C. for up to 24 hours prior to transfusion. For each unit processed, the in vitro loss of cellular hemoglobin ( per cent) and the total supernatant hemoglobin (milligrams *International sachusetts.
Equipment
Company,
Needham,
Mas-
per unit) were measured as previously described.“3* 21 Cultures on blood agar, thioglycollate, and Sabouraud’s medium were made routinely with each unit. Even though the preserved erythrocytes were transfused to the original donor, compatibility testing was performed in all cases using antiglobulin, albumin, and saline techniques, together with ABO and Rh typing. Autologous blood replacement. At the time of operation, blood loss was estimated from the measured suction volume and the weight of the sponges used. The two units of autologous previously frozen red cells was transfused, usually during the operation and always within the first 24 hour postoperative period. For up to six days following the surgical period serial hematocrit measurements were obtained on patients in the three groups. Groups A and B received autologous blood and Group C received no blood transfusion. Results The 11 patients in Group A were bled 21 units and 17 of these were transfused while the 12 patients in Group B were bled 22 units and 20 of these transfused. The six of the total 43 units was not administered because of lack of clinical indications. The transfusions were free of any untoward side effects, i.e., hemoglobinuria, jaundice, fever, chills, or urticaria. Figs. 1 and 2 show the red cell volumes and total cellular hemoglobin levels in Groups A and B prior to the two bleedings and prior to the operation. Both red cell volume and total cellular hemoglobin mass were significantly reduced when the two bleedings were separated by a two-week interval, but not when the bleedings occurred four weeks apart. Table II shows the statistical analysesI” of the results of the bleeding schedules. All units of previously frozen autologons blood were sterile and serologically compatible. The mean in vitro loss related to tke freeze preservation was 22.9 per cent, with a S.D. of k3.78, and a range of 16.7 to 32.0
Volume Number
Autotransfusions
105 3
of frozen
blood
397
Group A BLED,5ODml
BLED, 500 ml
i
-
!zig
0
-8
SURGERY
8’
4
2
-
6
8
S,URGERY
BLED, 500 ml
BLED, 500 ml 1750T11
l-l’
0
4
2 WEEKS
FOLLOWING
6 INITIAL
8
BLEED
Fig. 1. The mean red cell volumes for the patients in Groups A and B immediately the first and second bleedings and on the day before elective surgery. The intervals the first bleeding, the second bleeding, and the elective surgery are shown.
prior to between
Group ABLED,500 ml
?URGERY
BLED, 500 ml
54 ; ~~ED,500m~i’,~-;
f=y 8
4
0 WEEKS
FOLLOWING
INITIAL
BLEED
Fig. 2. The mean total cellular hemoglobin levels for the patients in Groups A and B immediately prior to the first and second bleedings and on the day before elective surgery. The intervals between the first bleeding, the second bleeding, and the elective surgery are shown. per cent. The mean total amount of supernatant hemoglobin per unit was 501 mg., with ii SD. of +327, and a range of 199 to 1,208 mg. Table III reports the estimated blood loss and the preoperative and five- to six-day
postoperative hematocrits in the three groups. A significant reduction in hematocrit occurred in the ten patients who did not receive any transfusions (Group C) . The mean estimated blood loss was similar in each of the three groups.
398
Daane,
Valeri,
and
October 1, 1969 Am. .I. Obst. & Gyocc.
Barton
Table II. Paired comparisons of red cell volumes, total cellular and peripheral hematocrits in Groups A and B* Initial-second
Group Means
A (two-week t S.D. t
bleeding
Total cellular hemoglobin level (cm.)
Red cell volume (ml.) interval) -104.2 t 11.6 2.39
hemoglobin
-38.1
? 6.7 2.48
levels,
Initial-jreoperative Red cell volume (ml.)
Hematocrit (vol. %) -1.3
t 1.4 1.82
-61.6
.l_____l
Total cellular hemoglobin level (Gm.1
+ 13.2 1.06
-19.3
Hematocrit (vol. To)
i: 8.2 0.85
-0.7
n go5
P Group Means
*With
B (four-week ? S.D. t n P
$05
2 9.1 0.82 zz.2
use of the t test for paired
to six-day
+ 5.6
2 6.3 0.16
-0.7
::.5
>09.2
2 1.1 1.16 2.06
+78.3
I? 8.6 3.11 <09.02
+14.6
f 5.3 1.52 >09.05
+0.3
? 1.4 0.33 9 >0.5
observations.‘3
mean blood loss and paired postoperative hematocrits Mean
Groups
>90.2
interval) +20.4
Table III. Estimated five-
iY.1
f. 1.7 0.31 9 >0.5
estimated blood loss (C.C.)
comparisons
Preoperative hematocrits
of preoperative
and the
Postoperative hematocrits
Mean difference
Group
A (n =
10)
485
38.1
36.2
-1.9 t =
+ 1.7 1.79 p > 0.2
Group
B (n =
11)
670
40.1
37.8
-2.3 t =
Group
C (n =
10)
420
40.8
34.1
p
Comments The transfusion of autologous red ceils is the ideal form of supportive hemotherapy for patients undergoing elective operations. The data in Table III indicate that the gynecologic surgery produced a significant decrease in peripheral hematocrit level in the postoperative period in the patients who were not transfused (Group C) . Patients in Groups A and B, who received two units of autologous blood, did not show this significant decrease. The results indicate that it is preferable for normalization of total red cell volume and cellular hemoglobin to utilize a fourweek interval in the bleeding-surgery sched-
>
-6.7 t = p
rt 1.8 2.23
<
0.05
t
1.7 7.69 0.001
ule rather than only two weeks. These findings corroborate those of Wadsworth who measured the mean hemoglobin concentration following 400 ml. venesections in healthy male and female subjects: the hemoglobin levels were lowest one or two weeks after bleeding and thereafter rapidly increased, reaching predonation levels three to four weeks after bleeding.27 In our study, measurements of red cell volume (milliliters) and mean corpuscular hemoglobin concetl; tration (grams per 100 ml.) characterize the total circulating cellular hemoglobin, which is the major determinant of the patient’s oxygen-carrying capacity at the time of operation.
Volume Number
Autotransfusions
105 3
Several investigators have shown that the collection and preservation of blood in liquid media for planned autotransfusion necessitates frequent and complicated bleeding schedules.3l 7l I19I8 Total cellular hemoglobin levels and red cell volumes in the patients studied by these investigators were not aeported; hematocrit, hemoglobin concentration, and reticulocyte values are percentage measurements and accurately reflect neither the total volume of red cells nor total cellular hemoglobin level. Freeze-preservation techniques which afford practical storage in the frozen state for two years permit a more versatile and flexible bleeding schedule for the collection of the autola’gous blood. 26 To be acceptable,
freeze-preservation technique must provide a clinically satisfactory biologic product of proved therapeutic effectiveness; the Huggins
method
of red cell
preservation
meets
26 However, the in this requirement. *I 22-241 vitro loss of 23 per cent, the 6.7 L. of wash
of frozen
blood
399
solution required, and the time involved in the preparation of a unit (50 minutes) indicate that this method is less than ideal. Freeze preservation of human red cells has progressed beyond the experimental phase to an advanced developmental phase.
Recent improvements in methods for washing glycerolized red cells are sufficiently encouraging to suggest that will soon become available.
a better
Even with autotransfusions,
system
however, the
risks of circulatory overload, microbial contamination, and inadvertent misadministration of the blood unit remain, and these hazards logically should continue to influence the clinical decision for or against
transfusion. The authors would like to acknowledge the excellent technical assistance of Miss Sally Gildea and Kenneth Patti, HM3, USN. The authors would also like to acknowledge with gratitude the critical review of this manuscript by Dr. Joseph G. Fink.
MfERENCES
1. &kerblom, O., deverdier, C. H., Finnson, M., Garby, L., HBgman, C. F., and Johansson, ti. G. 0.: Transfusion 7: 1, 1967. 2. Almond, D. V., and Valeri, C. R.: Transfusion 7: 95. 1967. 3. Ascari, W. Q., Jolly, P. C., and Thomas, P. A.: Transfusion 8: 111. 1968. 4. Aster, R. H., and Jan& J. H.: J. Clin. Invest. 43: 856, 1964. 5. Brittingham, T. E., and Chaplin, H., Jr.: T. A. M. A. 165: 819. 1957. 6. &by, W. H., M&n, J. I., and Furth, F. W.: U. S. Armed Forces M. J. 5: 693, 1954. 7. Cuello, L., Vazquez, E., PCrez, V., and Raffuc:ci, F. L.: Transfusion 7: 309, 1967. 8. Dausset, J.: Compt. rend. Sot. biol. (Paris) 148: 1607, 1954. 9. Gerst, P. ‘H., Horowitz, H. I., Rowe, A. W., Allen, F. H., and Kellner, A.: New York Sta.te J. Med. 67: 830, 1967. 10. Goudsmit, R., and van Loghem, J. J.: VOX. Sang. 3: 58, 1953. 11. Grant, F.: Ann. Surg. 74: 253, 1961. 12. Gray, S. J., and Sterling, K.: J. Clin. Invest. 29: 1604, 1950. 13. Hays, W. L.: Statistics for psychologists, New York, 1963, Holt, Rinehart & Winston, pp. 333-335. 14. Huggins, C. E.: Science 139: 504, 1963.
15. Huggins, C. E.: Monogr. S. SC. 3: 133, 1966. 16. Jones, A. R.: New England J. Med. 254: 172, 1956. 17. Marchal, G., Dausset, J., and Colombani, J.: 319-323 Comm. 8th Cong. Internat. Sot. Blood Transf., Tokyo, Japan. 18. Milles, G., Langston, H., and Dalessandro, W.: Surg. Gynec. & Obst. 115: 689, 1962. 19. Mollison, P. L.: Blood transfusion in clinical medicine, ed. 4, Philadelphia, 1967, F. A. Davis Company, pp. 590-641. 20. Moss, G. S., Valeri, C. R., and Brodine, C. E.: New England J. Med. 278: 747, 1968. 21. Shields, C. E.: Transfusion 8: 1, 1968. 22. Szymanski, I. O., and Valeri, C. R.: Transfusion 8: 74, 1968. 23. Valeri, C. R., and Bond, J. C.: Transfusion 6: 254, 1966. 24. Valeri, C. R., Runck, A. H., and McCallum, L. E.: Transfusion 7: 105, 1967. 25. Valeri, C. R.: Transfusion 8: 210, 1968. 26. Valeri, C. R., and Runcky, A. H.: Transfusion 9: 5, 1969. 27. Wadsworth, G. R.: J. Physiol. 129: 583, 1955. 28. Wallace, J. M., and Henry, J. B.: Transfusion 5: 153. 1965. 29. Wilson; J. D., and Taswell, H. F.: Mayo Clin. Proc. 43: 26, 1968.
of previously frozen blood
in elective gynecologic surgery THOMAS C.
A.
ROBERT
ROBERT Chetsea,
K.
DAANE,
COMMANDER
(MC)
VALERI,
COMMANDER
BARTON,
CAPTAIN
(MC) (MC)
USN* USNR USN**
Massachusetts
Autotransfusions of previously froten red cells in patients undergoing elective gynecotogic surgery are reported. When red cell volume and total circulating hemoglobin levels are used as criteria, a 4 week interval between the initial and second bleed proved better than a 2 week interval for the collection of two units of blood. The clinical responses to the autotogous transfusions were satisfactory.
blood is limited by its relatively short shelflife : acid-citrate-dextrose (ACD) collected blood can be satisfactorily stored at 4“ C. for no more than 21 days, while adenine supplementation of the ACD medium (AGDadenine) extends the storage period at 4’ C. up to 35 days. *I 21 Preservation of human red cells by freezing permits storage for prolonged periods and thereby lends itself to less rigidity in the scheduling of the bleeding and the surgery. This study was undertaken in order to evaluate the feasibility of a freeze-preservation approach to providing autologous blood for elective gynecologic surgery. At the same time, an attempt was made to ascertain the optimum bleeding schedule for collection of two units of blood prior to elective surgery. The red cells were preserved with highconcentration glycerol and the slow freezethaw technique. The red cells were washed by the Huggins technique, which entails dilution of the glycerolized red cells with nonelectrolyte solutions and recovery by agglomeration, I41 I5 Previous extensive clinical evaluation of preserved red cells by means of small aliquot autotransfusions to healthy volunteers, multiple (two or three) unit homologous transfusions to stable, anemic recipients, and multiple (ten or more)
THEUSEOFHOMOLOGOUS compatible blood to replace surgical blood loss is attended by two major hazards: isosensitization to red cells, white cells, or platelets, and the transmission of disease, particularly serum hepatitis and malaria.4p 5l 8* lo, 171 I91 28 To circumvent these difficulties, autologous blood transfusion has been frequently recommended for elective surgery.3* 7* g, I19 I81 20 For this purpose, the use of liquid preserved
From the Department of Obstetrics and Gynecology, Naval Hospital, and the Naval Blood Research Laboratory. This work was supported by the U. S. Navy. The opinions or assertions contained herein are those of the authors, and are not to be construed as oficial or reflecting the views of the Navy Department or the Naval Service at large. This paper was presented in part at the Armed Forces District Meeting, American College of Obstetrics and Gynecology, Las Vegas, Nevada, Oct. 23, 1967, and District Z Meeting, American College of Obstetrics and Gynecology, Boston, Massachusetts, Oct. 25, 1967. ‘Present address: Department Obstetrics and Gynecology, of California at Los Angeles, Angeles, California 90024. **Present address: Military Assistance APO San Francisco,
Headquarters, Command, Californta
of University Los U. S. Vietnam, 96222.
394
Volume
105
Number
3
Autotransfusions
Table I. Diagnoses of the patients studied in three groups G7OU
4
No.
Group A-11 patients (two-week bleedings) Mean age, 38.3 yr. Mean parity, 4.6 Pelvic relaxation Myoma of uterus Carcinoma in situ of cervix Ch.ronic pelvic inflammatory disease Group .I-12 patients (four-week bleedings) Mean age, 38.6 yr. Mean parity, 4.5 Pelvic relaxation Myoma of uterus Carcinoma in situ of cervix Control group-10 patients (no Mean age, 37.2 yr. Mean parity 3.8 Pelvic relaxation Myoma of uterus Carcinoma in situ of cervix Chronic pelvic inflammatory disease
interval
between
5 3 2 1 interval
between
8 2 2 bleedings)
5 3 1 1
homologous transfusions to severely wounded servicemen in South Vietnam, showed this blood to be safe and efficacious.2* 2ol22-24 Mat,erials
and
methods
Selection and grouping of patients. All of the patients were candidates for elective gynecologic surgery. Table I details the mean age, mean parity, and diagnoses of the patients in each of three groups. Patients in Groups A and B were bled on two occasions. On each occasion approximately !WO ml. of whole blood was collected in 75 ml. ACD (National Institutes of Health, Formula A). In Group A there was a 2 week interval between the two bleedings, whereas in Group B 4 weeks intervened between bleedings. In both groups the elective surgery was performed 4 weeks after the second bleed. The patients in Group C were not bled, serving as the controls for the evaluation of pre- and postoperative hematocrits in patients in Groups A and B undergoing surgery for similar disorders. All of the patients in Groups A and B received
of frozen
blood
395
a high protein diet and 150 Gm. of ferrous fumarate* (approximately 50 mg. of elemental iron) twice daily following the first bleeding. The patients with pelvic relaxation had vaginal hysterectomy and/or culpoplasty and/or culpoperineoplasty; those with leiomyomata uteri, carcinoma in situ of the cervix, and chronic pelvic inflammatory disease had abdominal hysterectomy with or without removal of the adnexa. Red cell volume measurements. Chromium-51 red cell volumes were measured in each patient in Groups A and B immediately prior to the first and second bleedings, and on the day before the elective operation. At the time of the first measurement, 20 ml. of whole blood was collected in 3.0 ml. of modified ACD medium.t Five microcuries (PC) of Naz5*Cr0,$ was added and the blood was incubated at 37O C. for 30 minutes.12 Ascorbic acid was not added nor were the labeled erythrocytes washed.25 A 10 ml. aliquot of the labeled red cells was injected into the patient and a sample collected 30 minutes later. The red cell volume was then reckoned from the observed dilution of the injected red cells’ radioactivity. The red cell volume was measured the second time with 12 to 15 PC of 51Cr, and the third time with 25 ,.LCof 51Cr. A correction for radioactivity in the recipient red cells was required for the second and third red cell volume measurements. The mean corpuscular hemoglobin concentration (MCHC), measured in grams per cent, was derived from the microhematocrit and the total hemoglobin concentration, measured on the day of the red cell volume determination (in milliliters) .61I6 The total cellular hemoglobin level was calculated by multiplying the red cell volume by the MCHC. Red cell preservation. The ACD-collected blood was stored at 4O C. for up to five days. Each unit was then centrifuged in a PR-2 refrigerated centrifuges at 3,500 f 200 *Ferrosequels,
Lederle
Laboratories.
iSquibb ACD solution mod&d (IO ml.) containing citric acid 80 mg., sodium citrate 250 mg., and dextrose anhydrous 120 mg. &Squibb
Chromotope.
$International sachusetts.
Equipment
Company,
Needham,
Mas-
396
Daane,
Valeri,
and Barton
October 1, 1969 Am. J, Obst. &Gym.
r.p.m. for six minutes, following which the plasma was removed and stored at -20° C. The packed red cell mass was transferred to a Blood Freezing Unit* and glycerolized in a Huggins Cytoglomerator* with an equal volume of 8.6M glycerol containing 0.3 per cent disodium ethylenediaminetetra-acetic acid (Na, EDTA), 8 per cent glucose, and 1 per cent fructose. The glycerolized red cell mass was slowly frozen, and stored at -80’ C. for up to two months. On the day of the elective operation each unit was thawed in the 40’ C. water bath of the Huggins Cytoglomerator for approximately five minutes. Each thawed, glycerolized red cell mass was diluted during continuous agitation, first with 500 ml. of 50 per cent dextrose in water, and then with approximately 2,000 ml. of a nonelectrolyte solution containing 8 per cent glucose and 1 per cent fructose. When agitation was stopped the red cells sedimented by spontaand the supernatant neous agglomeration, was expressed into the appropriate section of the Blood Freezing Unit. The red cells were washed twice more with two additional 2,000 ml. volumes of the nonelectrolyte solution, each time being recovered by spontaneous agglomeration when the agitation ceased. The agglomerated red cell mass was disaggregated with 250 ml. of isotonic saline solution. The disaggregated red cells were placed in the PR-2 refrigerated centrifuge and spun at 3,500 t 200 r.p.m. for six minutes, after which the supernatant fluid was removed completely and the thawed autologous plasma added. Approximately 50 minutes was required for the preparation of the two units of blood. The final hematocrit ranged from 25 to 49 with a mean of 32 per cent and a standard deviation of _+ 4.7. The previously frozen red cell mass resuspended in autologous plasma was then stored at 4O C. for up to 24 hours prior to transfusion. For each unit processed, the in vitro loss of cellular hemoglobin ( per cent) and the total supernatant hemoglobin (milligrams *International sachusetts.
Equipment
Company,
Needham,
Mas-
per unit) were measured as previously described.“3* 21 Cultures on blood agar, thioglycollate, and Sabouraud’s medium were made routinely with each unit. Even though the preserved erythrocytes were transfused to the original donor, compatibility testing was performed in all cases using antiglobulin, albumin, and saline techniques, together with ABO and Rh typing. Autologous blood replacement. At the time of operation, blood loss was estimated from the measured suction volume and the weight of the sponges used. The two units of autologous previously frozen red cells was transfused, usually during the operation and always within the first 24 hour postoperative period. For up to six days following the surgical period serial hematocrit measurements were obtained on patients in the three groups. Groups A and B received autologous blood and Group C received no blood transfusion. Results The 11 patients in Group A were bled 21 units and 17 of these were transfused while the 12 patients in Group B were bled 22 units and 20 of these transfused. The six of the total 43 units was not administered because of lack of clinical indications. The transfusions were free of any untoward side effects, i.e., hemoglobinuria, jaundice, fever, chills, or urticaria. Figs. 1 and 2 show the red cell volumes and total cellular hemoglobin levels in Groups A and B prior to the two bleedings and prior to the operation. Both red cell volume and total cellular hemoglobin mass were significantly reduced when the two bleedings were separated by a two-week interval, but not when the bleedings occurred four weeks apart. Table II shows the statistical analysesI” of the results of the bleeding schedules. All units of previously frozen autologons blood were sterile and serologically compatible. The mean in vitro loss related to tke freeze preservation was 22.9 per cent, with a S.D. of k3.78, and a range of 16.7 to 32.0
Volume Number
Autotransfusions
105 3
of frozen
blood
397
Group A BLED,5ODml
BLED, 500 ml
i
-
!zig
0
-8
SURGERY
8’
4
2
-
6
8
S,URGERY
BLED, 500 ml
BLED, 500 ml 1750T11
l-l’
0
4
2 WEEKS
FOLLOWING
6 INITIAL
8
BLEED
Fig. 1. The mean red cell volumes for the patients in Groups A and B immediately the first and second bleedings and on the day before elective surgery. The intervals the first bleeding, the second bleeding, and the elective surgery are shown.
prior to between
Group ABLED,500 ml
?URGERY
BLED, 500 ml
54 ; ~~ED,500m~i’,~-;
f=y 8
4
0 WEEKS
FOLLOWING
INITIAL
BLEED
Fig. 2. The mean total cellular hemoglobin levels for the patients in Groups A and B immediately prior to the first and second bleedings and on the day before elective surgery. The intervals between the first bleeding, the second bleeding, and the elective surgery are shown. per cent. The mean total amount of supernatant hemoglobin per unit was 501 mg., with ii SD. of +327, and a range of 199 to 1,208 mg. Table III reports the estimated blood loss and the preoperative and five- to six-day
postoperative hematocrits in the three groups. A significant reduction in hematocrit occurred in the ten patients who did not receive any transfusions (Group C) . The mean estimated blood loss was similar in each of the three groups.
398
Daane,
Valeri,
and
October 1, 1969 Am. .I. Obst. & Gyocc.
Barton
Table II. Paired comparisons of red cell volumes, total cellular and peripheral hematocrits in Groups A and B* Initial-second
Group Means
A (two-week t S.D. t
bleeding
Total cellular hemoglobin level (cm.)
Red cell volume (ml.) interval) -104.2 t 11.6 2.39
hemoglobin
-38.1
? 6.7 2.48
levels,
Initial-jreoperative Red cell volume (ml.)
Hematocrit (vol. %) -1.3
t 1.4 1.82
-61.6
.l_____l
Total cellular hemoglobin level (Gm.1
+ 13.2 1.06
-19.3
Hematocrit (vol. To)
i: 8.2 0.85
-0.7
n go5
P Group Means
*With
B (four-week ? S.D. t n P
$05
2 9.1 0.82 zz.2
use of the t test for paired
to six-day
+ 5.6
2 6.3 0.16
-0.7
::.5
>09.2
2 1.1 1.16 2.06
+78.3
I? 8.6 3.11 <09.02
+14.6
f 5.3 1.52 >09.05
+0.3
? 1.4 0.33 9 >0.5
observations.‘3
mean blood loss and paired postoperative hematocrits Mean
Groups
>90.2
interval) +20.4
Table III. Estimated five-
iY.1
f. 1.7 0.31 9 >0.5
estimated blood loss (C.C.)
comparisons
Preoperative hematocrits
of preoperative
and the
Postoperative hematocrits
Mean difference
Group
A (n =
10)
485
38.1
36.2
-1.9 t =
+ 1.7 1.79 p > 0.2
Group
B (n =
11)
670
40.1
37.8
-2.3 t =
Group
C (n =
10)
420
40.8
34.1
p
Comments The transfusion of autologous red ceils is the ideal form of supportive hemotherapy for patients undergoing elective operations. The data in Table III indicate that the gynecologic surgery produced a significant decrease in peripheral hematocrit level in the postoperative period in the patients who were not transfused (Group C) . Patients in Groups A and B, who received two units of autologous blood, did not show this significant decrease. The results indicate that it is preferable for normalization of total red cell volume and cellular hemoglobin to utilize a fourweek interval in the bleeding-surgery sched-
>
-6.7 t = p
rt 1.8 2.23
<
0.05
t
1.7 7.69 0.001
ule rather than only two weeks. These findings corroborate those of Wadsworth who measured the mean hemoglobin concentration following 400 ml. venesections in healthy male and female subjects: the hemoglobin levels were lowest one or two weeks after bleeding and thereafter rapidly increased, reaching predonation levels three to four weeks after bleeding.27 In our study, measurements of red cell volume (milliliters) and mean corpuscular hemoglobin concetl; tration (grams per 100 ml.) characterize the total circulating cellular hemoglobin, which is the major determinant of the patient’s oxygen-carrying capacity at the time of operation.
Volume Number
Autotransfusions
105 3
Several investigators have shown that the collection and preservation of blood in liquid media for planned autotransfusion necessitates frequent and complicated bleeding schedules.3l 7l I19I8 Total cellular hemoglobin levels and red cell volumes in the patients studied by these investigators were not aeported; hematocrit, hemoglobin concentration, and reticulocyte values are percentage measurements and accurately reflect neither the total volume of red cells nor total cellular hemoglobin level. Freeze-preservation techniques which afford practical storage in the frozen state for two years permit a more versatile and flexible bleeding schedule for the collection of the autola’gous blood. 26 To be acceptable,
freeze-preservation technique must provide a clinically satisfactory biologic product of proved therapeutic effectiveness; the Huggins
method
of red cell
preservation
meets
26 However, the in this requirement. *I 22-241 vitro loss of 23 per cent, the 6.7 L. of wash
of frozen
blood
399
solution required, and the time involved in the preparation of a unit (50 minutes) indicate that this method is less than ideal. Freeze preservation of human red cells has progressed beyond the experimental phase to an advanced developmental phase.
Recent improvements in methods for washing glycerolized red cells are sufficiently encouraging to suggest that will soon become available.
a better
Even with autotransfusions,
system
however, the
risks of circulatory overload, microbial contamination, and inadvertent misadministration of the blood unit remain, and these hazards logically should continue to influence the clinical decision for or against
transfusion. The authors would like to acknowledge the excellent technical assistance of Miss Sally Gildea and Kenneth Patti, HM3, USN. The authors would also like to acknowledge with gratitude the critical review of this manuscript by Dr. Joseph G. Fink.
MfERENCES
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15. Huggins, C. E.: Monogr. S. SC. 3: 133, 1966. 16. Jones, A. R.: New England J. Med. 254: 172, 1956. 17. Marchal, G., Dausset, J., and Colombani, J.: 319-323 Comm. 8th Cong. Internat. Sot. Blood Transf., Tokyo, Japan. 18. Milles, G., Langston, H., and Dalessandro, W.: Surg. Gynec. & Obst. 115: 689, 1962. 19. Mollison, P. L.: Blood transfusion in clinical medicine, ed. 4, Philadelphia, 1967, F. A. Davis Company, pp. 590-641. 20. Moss, G. S., Valeri, C. R., and Brodine, C. E.: New England J. Med. 278: 747, 1968. 21. Shields, C. E.: Transfusion 8: 1, 1968. 22. Szymanski, I. O., and Valeri, C. R.: Transfusion 8: 74, 1968. 23. Valeri, C. R., and Bond, J. C.: Transfusion 6: 254, 1966. 24. Valeri, C. R., Runck, A. H., and McCallum, L. E.: Transfusion 7: 105, 1967. 25. Valeri, C. R.: Transfusion 8: 210, 1968. 26. Valeri, C. R., and Runcky, A. H.: Transfusion 9: 5, 1969. 27. Wadsworth, G. R.: J. Physiol. 129: 583, 1955. 28. Wallace, J. M., and Henry, J. B.: Transfusion 5: 153. 1965. 29. Wilson; J. D., and Taswell, H. F.: Mayo Clin. Proc. 43: 26, 1968.