On highly speculative grounds, the postulate was made that monochloroacetate might itself have a potent antibacterial action. If so, the use of heat and formaldehyde for plasma sterilization could then be abandoned. Fortunately, monochloroacetate in concentrations of 0.02 thousand or 0.04 thousand per liter added to heavily contaminated human plasma displayed a surprisingly high degree of antibacterial activity. Some of the pathogens tested are shown in table 7.

When the appropriate culture medium for a particular organism was liquid in form, one part of the culture broth was added to nine parts of acetatetreated plasma. In each instance, the culture and its organisms were not added until bacterial growth was maximal; thus, bacterial contamination was unusually heavy for those samples of plasma tested. Subsequent to inoculation, aliquots of contaminated plasma were held at 3° C., 23° C., and 37° C. The results shown in table 7 are those obtained from contaminated acid-treated plasma held at 23° C.

Fortunately, monochloroacetate in the 0.02 thousand final concentration maintains its antibacterial action for at least 7 months, despite frequent intentional bacterial contaminations introduced during this protracted period of time. This observation suggests that a pool or unit of plasma, whether originally contaminated or not, should maintain its bacterial sterility in spite of subsequent breaking of its seal which would permit bacterial contamination. We have not yet examined for the possibility that bacterial resistance to monochloroacetate may develop.

A 8 HOURS @60°C

A2 HOURS @60°c 44 HOURS@60°c 46 HOURS @60c

FIG. 1. Showing electrophoretic patterns of heated normal plasma above. The lower four patterns represent changes in electrophoretic patterns in plasma containing 0.02 molar chloroacetate after heating 2, 4, 6, and 8 hours respectively.

Paper electrophoresis (Spinco) was performed upon plasma containing monochloroacetate. The products examined were aged for varying periods of time with and without the presence of acetate. The electrophoretic results obtained disclose little if any change which did not also occur in the aliquot of citrated plasma derived from the same pool when monochloroacetate was not added, provided both the treated and untreated samples were of the same age and had been maintained at similar temperatures (fig. 1).

Measurements of plasma viscosity and optical density disclosed no demonstrable differences between ordinary plasma and that to which 0.02 or 0.04 thousand had been added.

The biologic tolerance for acetate-treated plasma was evaluated by the daily administration of canine plasma to dogs. The volumes of treated plasma given each day were equal to 50 percent of the estimated plasma volume of the animal. These quantities of plasma were administered daily for periods of time ranging from 28 to 42 days. The animals were maintained on a nonprotein diet containing adequate minerals and vitamins throughout their test periods. The only source of protein available was that contained in the infused plasma. Both the volume of plasma and the total allowed calories were determined on the basis of body weight. To compare the relative merits of acetate-treated plasma against those of an aliquot of ordinary citrated plasma derived from a common pool, half of the animals received plasma containing 0.02 thousand of monochloroacetate per liter and the others, serving as controls, receive citrated plasma without acetate.

The results of plasma infusions in both groups were identical throughout the entire study period. The animals remained afebrile, maintained their body weight, and were sustained in positive nitrogen balance. Other studies included erythrocyte, leukocyte, and thrombocyte counts on arterial blood and blood smears; hemoglobin determinations, bone marrow biopsy, prothrombin and whole blood clotting time, alkaline phosphatase and acid phosphatase activities, serum bilirubin, nonprotein nitrogen, and electrophoretic determinations. The results from these tests also disclosed no detectable difference between those animals receiving ordinary citrated plasma and those whose plasma also contained monochloroacetate. The same tests were continued following the completion of the infusion periods and no latent abnormalities thus far have been detected.

Biopsies performed on lung, liver, spleen, gut, adrenal glands, and the kidneys at the end of the infusion period in each animal disclosed no histologic abnormality.

Because we observed no evidence of toxicity from acetate-treated plasma in our fairly numerous experimental studies, it seemed appropriate to explore the therapeutic potential of human plasma to which monochloroacetate had been added to give a final concentration of 0.02 thousand or 0.04 thousand per liter of plasma. In each instance, the pooled plasma used had been aged for 10 weeks at 38° C. to minimize the risk of transmitting serum hepatitis prior to the addition of acetate. Thereafter the product was administered within a few days, 550 milliliters to the unit.

To date, 11 debilitated patients suffering from carcinomatosis have received from 3 to 8 units of this plasma which they tolerated without detectable reactions or latent ill effects. Their general responses were those of improvement to the extent this was possible in patients suffering from a fatal illness. Laboratory studies, save for nitrogen balance and organ biopsies, were similar to those performed under the aforementioned experimental conditions. No abnormalities were detected. Two patients subsequently have died of their malignant disease; histologic examination of their tissues were normal, except for widespread carcinomatosis.

The possiiblity that acetate-treated plasma might prove to be allergenic cannot be answered. However, in two patients the administrations of plasma were separated by 2- and 4-week intervals without evidence of untoward reactions of any kind.

A series of in vitro studies, similar to those of plasma, were carried out on whole blood. The antibacterial action of monochloroacetate was just as effective for blood as was observed in plasma. The organisms tested were the same as those added to plasma; the intensity of concentration of the bacterial culture added to blood was comparable to that employed in the studies on plasma.

Other studies on blood included the comparisons of acetate-treated blood with that for untreated aliquots from the same sample of citrated human blood.

Among the qualities tested were red-cell fragility, their oxygen-carrying capacity, the lifespan of chromium-tagged red cells, hemoglobin diffusion into plasma when treated and untreated blood were allowed to age for 22 days at 3° C., spectrographic analyses for detection of abnormal hemoglobin pigment, and the concentrations of potassium in the supernatant plasma of the acetate-treated and the normal citrated samples derived from the same unit of blood. In each instance, no discernible difference was observed in the results obtained. Stated otherwise, these studies disclosed no harmful effects to whole blood when monochloroacetate had been added. Further studies are necessary to comment upon the toxicity following the transfusion of blood containing monochloroacetate, though in the limited extent to which these studies have been carried out, no toxic manifestations have been observed. A detailed report of these studies will be presented elsewhere.

DISCUSSION

The serious problem of hepatitis from whole blood transfusions goes unacknowledged largely because there is no means whereby this complication can be anticipated, prevented, or treated. Further, its magnitude is partially obscured by the difficulty in tracing patients after transfusions. Thus it is necessary to accept serum hepatitis as a complication of whole blood, a calculated risk to be considered whenever blood is to be transfused. The magnitude of this risk, however, has not been generally appreciated.

The mortality in our patients transfused with blood without plasma was approximately 1 death from serum hepatitis for every 500 patients receiving transfusions of whole blood, a transfusion mortality from this cause alone which approximates that of appendectomy for nonperforative acute appendicitis. The overall mortality from serum hepatitis in our series was 13.9 percent. As pointed out earlier, the attack rate for hepatitis from blood transfusions in our patients may be higher than those experienced by others who need not rely so heavily upon the use of professional donors.

Risks, however, must be weighed against gains in any form of therapy. Thus, there is no question that blood replacement is imperative to the successful treatment of patients in shock due to losses of blood or plasma. Moreover, there is substantial clinical evidence to support the thesis that most patients recover more rapidly if normal blood volume is maintained. Further, the incidence of fatal postoperative pulmonary embolism and of acute renal failure appears to be greater in patients in whom shock occurs and is inadequately treated during surgical operations or following trauma. To withhold blood when clearly needed, simply for the fear of transmitting serum hepatitis, cannot possibly be justified. However, the question should be asked: How important is a single 500-ml. unit of blood to the life of an adult patient if this is all that was necessary to restore his circulatory status? Would not another fluid or drug have served almost as well? Unfortunately, one does not always know that only one transfusion is needed; hence, it is often better to start a transfusion rather than to allow the patient's condition to deteriorate further. Thirty-six percent of all patients transfused in this series received only one unit of blood; since the value of the single transfusion has been questioned in this institution for many years, it is our hope that this 36 percent represents a smaller percentage of patients receiving single transfusions than may previously have obtained. Most transfusions given are probably necessary at the time they are given, but it also seems clear that some were given which may not have been required, at least in retrospect. The potent antibacterial action of 0.02 M or 0.04 M monochloroacetate in blood or plasma and its low degree of toxicity appears to warrant further exploration in both blood and plasma with the view to its possible routine use in the preparation of plasma transfusions as a possible means for the prevention of bacterial growth should contamination occur unwittingly in the course of its preparation. If the observations herein reported be confirmed by others, the use of monochloroacetate may simplify many of the more cumbersome steps in plasma manufacture, especially those which concern its continuing bacterial sterility. Though the antibacterial action of monochloroacetate proved potent, the reasons which led us to postulate its use in this respect is open to serious question.

Further studies on toxicity of blood transfusions containing monochloroacetate are necessary before attempting a study on the use of this agent as an additive for clinical use in blood transfusions. The need for such an agent is selfevident and, if proven effective and safe, its use would permit the return of unused blood to the blood bank for reissue, thereby conserving blood, especially

that taken to the operating room, as a precautionary measure in anticipation of a need which does not materialize. Present losses of blood incurred in those institutions performing surgery, requiring the use of some form of extracorporeal circulation, have been especially distressing. A method which allowed the recovery and reissue of units of blood held at 37° C. for several hours, in case of need during the course of open-heart surgery, would relieve a pressing problem in blood supply.

Several years ago Dr. Charles B. Huggins suggested to one of us (J.G.A.) the possibility that bromoacetate or iodoacetate might possess virocidal activity. At that time neither of us was aware of the antibacterial activity of monochloroacetate; we do not yet know that this agent has antiviral activity, though we are actively engaged in such a study at this time.

SUMMARY

1. Thirty-six established cases of serum hepatitis occurred among a randomized selected series of 2,388 patients, 1,894 of whom survived at least 6 months after their last blood transfusion; 8 other patients developed jaundice in which the diagnosis of serum hepatitis was held in question. An average of 3 units of blood was given each of the 2,388 patients in the series. Plasma or other icterogenic derivatives of blood had not been given; 5 of the 36 patients died.; and 2 of the 8 patients, in which the diagnosis was questionable, died.

(a) Blood donor sources in this series were of two classifications: family donors (friends and relatives of the patient), and professional donors. (The incidence of serum hepatitis with jaundice from professional sources was 7 to 10 times that experienced when all blood given was from family donors.). For those patients who developed serum hepatitis, when only 1 unit of blood was given, the attack rate was 0.3 percent; whereas, that of the single-unit transfusions was 3.2 percent when the unit was from a professional donor.

(b) A total of 189 cases of serum hepatitis are known to have developed among the total of 11,382 patients tallied as receiving blood only. These figures include the 2,388 patients in the randomized selection. Since only 21 percent of 2,388 patients of the total patients receiving blood without plasma were fully followed until death, or for at least 6 months after their last transfusion episode, the 145 cases which were known to have occurred among the nonstudy group, an additional 8,993 patients, it is probable that more cases of serum hepatitis could be detected in the nonstudied group were this group to be reviewed in its entirety. The total of 189 cases includes known cases developing through April 1, 1958, a period of time which was 15 months longer than the 101⁄2 years covered in the randomized sample.

(c) The mortality rate for serum hepatitis in the 36 patients in whom this diagnosis was established, among 1,894 patients receiving blood who survived 6 months, was 13.9 percent.

2. Data reported on the antibacterial (bactericidal) activity of the sodium salt of monochloroacetic acid when added as a 1-molar solution to citrated blood or plasma so that its final concentration was 0.02 M or 0.04 M per liter of plasma or blood proved successful in eradicating heavy bacterial contamination with human pathogens which included vegetative and nonvegetative forms as well as the tuberculosis bacillus.

(a) Surprisingly, plasma to which this acetate had been added displayed little if any detectable change in its electrophoretic pattern, viscosity, or optical density.

(b) Acetate-treated plasma was transfused to dogs in daily volumes equal to 50 percent of the animal's estimated plasma volume for periods of 28 to 42 days. No abnormal physiologic reactions or pathologic effects were observed. In no way was the animal's response to transfusions of acetate-treated plasma different from that of ordinary citrated plasma without the monochloroacetate additive. To date, 11 debilitated patients with advanced carcinomatosis have received a total of 53 units (550 ml. each) of pooled plasma containing 0.02 thousand or 0.04 thousand of acetate; all were improved clinically to the extent expected from the administration of similar volumes of ordinary plasma.

(e) Citrated whole blood to which monochloroacetate had been added (human and canine) gave no evidence that the lifespan of red cells was shortened; that the oxygen-carrying capacity of erythrocytes was altered; that red-cell fragility was increased; or that abnormal hemoglobin pigments developed. There was no evidence to suggest that citrated human blood containing this halogenated

acetate was different from aliquots of the same units of blood serving as controls for the 22-day period covered by these in vitro studies; both the test and the control aliquots were maintained at 3° C. Although animals have been transfused freely with homologous canine blood containing 0.02 thousand or 0.04 thousand concentration of the acetate per liter, o deleterious effects have been noted, but data on the tolerance of blood transfusions are insufficient at this time to warrant comment upon the possible use of this additive to human transfusions.

(d) The mode of action of this antibacterial agent has not yet been established. (e) We do not yet know if this agent has an antiviral propensity.

ACKNOWLEDGMENT

The authors are especially indebted to Mr. Kenneth Smith for his technical assistance on this project.

BIBLIOGRAPHY

1. Al-Shamma, A., W. W. Pfaff, E. A. Stemmer, and J. G. Allen: "Changes in Human Citrated Plasma Produced by 60° C. Heat Exposure for 10 Hours and the Protective Action of Formaldehyde and Dextrose." Surgical Forum, 1958, p. 91.

2. Gordon, J. E.: cited by Sawyer et al. (see below).

3. Jensen, E. V.: "The effects of pH. and of Sulhydryl Reagents on the Nature of the Coagulum." (J. of Biol. Chem., 185: 410, 1950).

4. Jervis, J. J.: quoted in "Memorandum Prepared by Medical Officers of the Ministry of Health," Lancet, 1: 83, 1943.

5. McNalty, A. S.: "Acute Infective Jaundice and Administration of Measles Serum. Annual Report of the Chief Medical Officer of the Ministry of Health for the Year 1937." London, His Majesty's Stationary Office, 1938 pp. 38–39.)

6. Murray, R., F. Ratner, W. C. L. Diefenbach, and H. Geller: "Effect of Storage at Room Temperature on Infectivity of Icterogenic Plasma." (JAMA, 155: 13, 1954.)

7. Murray, R., W. C. L. Diefenbach, F. Ratner, N. C. Leone, and J. W. Oliphant: "Carriers of Hepatitis Virus in the Blood and Viral Hepatitis in Whole Blood Recipients. 2. Confirmation of Carrier State by Transmission Experiments in Volunteers." (JAMA, 154: 1072, 1954.)

8. Pfaff, W. W., E. A. Stemmer, A. Al-Shamma, D. Dawson, and J. G. Allen: "The Treatment of Pooled Human Plasma With Formaldehyde at 60° C. for 10 Hours." (Surgical Forum, 1958, p. 95.)

9. Propert, S. A.: "Hepatitis After Prophylactic Serum." (Brit. Med. J., 2: 677, 1938.)

10. Sawyer, W. A., K. F. Meyer, M. D. Eaton, J. H. Bauer, P. Putnam, and F .F. Schwentker: "Jaundice in Army Personnel in the Western Region of the United States and Its Relation to Vaccination Against Yellow Fever, Parts II, III, and IV." (Amer. J. Hygiene, 40: 35, 1944.)

11. Sayman, W. A., R. L. Gauld, S. A. Star, and J. G. Allen: "Safety of Liquid Plasma-A Statistical Appraisal." (JAMA, 168: 1735, 1958.)

DISCUSSION

Dr. PAUL HOXWORTH. Mr. Chairman, members and guests, there are thousands of cases of serum hepatitis produced by blood transfusion each year. It is a serious disease, not only from the standpoint of the mortality which Dr. Allen expressed, but there is a serious incidence of chronic hepatitis and cirrhosis which often follows recovery from the acute phase.

Dr. Allen's paper is of importance to surgeons because they are the big users of blood, and as such should consider the risk and reduce the transmission as much as they can until a practicable method for eliminating viral activity is discovered.

(Slide.) Dr. Allen's 44 cases of hepatitis from 5,000-odd units of whole blood, an incidence of infectivity of 0.728, means that 1 unit in 127 transmitted active disease to the recipient. This should cause the perfusionists to think.

Our experience of 19 cases from 7,135 units, an incidence of infectivity of 0.259 percent, means that 1 unit in each 386 transmitted a disease. When projected, the difference in these figures would represent the difference between 40,000 and 12,000 cases of serum hepatitis each year in the United States.

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