HEMOGLOBIN CONTENT OF PLASMA OF BANKED BLOOD, AND OF SERUM OF PATIENTS WITH TRANSFUSION REACTIONS*

Transcription

1 HEMOGLOBIN CONTENT OF PLASMA OF BANKED, AND OF SERUM OF PATIENTS WITH TRANSFUSION REACTIONS* MORTON GROVJE-RASMUSSEN, M.D., AND LAMAR SOUTTER, M.D. From the Blood Bank of the Massachusetts General Hospital, Boston, Massachusetts I. CONCENTRATION OF HEMOGLOBIN IN PLASMA OF BANKED Some writers have reported the administration of hemoglobin.intravenously in considerable quantities to patients without apparent harmful effects. Muether and Andrews safely administered blood containing 00 to 00 mg. per 00 ml. Others have given solutions containing from to 0 grams of hemoglobin with few reactions,, most of which consisted of chills, fever and abdominal cramps. The liberation of hemoglobin from the erythrocytes not only reduces the oxygencarrying power of the blood but renders it less useful and possibly toxic. Although the injection of hemoglobin may be harmful, it seems impossible to state at the present time what a safe level of free hemoglobin is for any individual patient. Besides individual variations of kidney function, kidney reserve and sensitivity to hemoglobin and its breakdown products, there is the additional factor of the amount of blood received and the rapidity of its administration. Multiple transfusions are now commonly given. In shock due to hemorrhage transfusions are frequently given with great speed. As many as ten or fifteen transfusions may be administered within a four-hour period. Under these circumstances, therefore, it may be necessary to reduce our estimates of the safe level of free hemoglobin in banked blood. As a preliminary to a study of transfusion reactions, we considered it necessary to determine the mean and maximum values of free hemoglobin under usual conditions in our banked blood. One hundred flasks of blood were selected at random, the age of the blood being representative of specimens of blood that are usually given out. The supernatant plasma from 0 of these was examined for the presence of free hemoglobin; the same 0 were then examined for total free hemoglobin after resuspension of the cells. This first group and another 0 were then studied after filtering to determine the hemoglobin level just before the bloods were dispensed. The recipients of transfusions were then studied to learn if the number of reactions could be correlated with the amount of free hemoglobin in the blood they received. The quantitative determinations of hemoglobin were carried out as previously described. Certain factors in the collection, preservation and distribution of the blood should be mentioned as influencing the amount of hemolysis. Our blood is collected in Fenwal transfusion bottles using a -gauge needle and a closed gravity system. Agitation on collection is gentle. Seventy-five ml. of ACD No. solution is used as the anticoagulant. The bottles are kept at room temperature prior to collection of blood; thereafter they are immediately placed in a refrigerator. The refrigerator temperature is thermostatically controlled to remain within one * Received for publication, December,.. Downloaded from on 0 January 0

2 HEMOGLOBIN IN PLASMA AND SERUM and one-half degrees of C. The compressors are kept separate to avoid agitation. During storage, the blood flasks are moved on three different occasions from one compartment or refrigerator to another. These transfers take but a few seconds, are made by hand, and do not entail perceptible warming. Because of difficulty with Baxter and Cutter filters in the intravenous tubing between the flask of blood and the patient, prefiltering through stainless steel sieves of 0. mm. mesh was adopted in." Bloods so filtered are used immediately and may not be returned if unused. Many bloods that have produced reactions have been cultured but rarely has a specimen of blood yielded growth of organisms. These filters must be moistened with sterile saline to avoid surface tension which would prevent the passage of blood in some instances. There are several variables that influence the observed quantity of free hemoglobin. One is introduced by the method described previously which has an error up to per cent when the amount to be measured is small. Another is that the amount of blood collected varies from about to ml. per transfusion flask. Lastly, the individual ratios of cells to supernatant plasma vary considerably from flask to flask. It is interesting to speculate as to whether the amount of free hemoglobin in the supernatant plasma of a flask of blood that is allowed to settle for several days is representative of the amount in the blood as a whole. It is difficult to visualize a constant diffusion of hemoglobin as it is released from the cells. However, any measurement of the total hemoglobin made after resuspension of the cells includes some hemoglobin freed by agitation and some by centrifuging afterwards. In Table are recorded the amounts of free hemoglobin in the supernatant plasma in each of 0 flasks of banked blood. These samples were centrifuged at 00 r.p.m. for five minutes to precipitate any red cells, after which the plasma was re-aspirated. The hemoglobin content of the plasma from these bloods (average age of blood,. days) was. mg. per 00 ml. These same 0 bloods were gently agitated to resuspend the cells and, at the same time, to redistribute the free hemoglobin throughout the entire blood. Samples were then drawn off. These were centrifuged for five minutes, the plasma drawn off and recentrifuged, then aspirated for study. The results of these observations are set forth in Table. As will be seen, the hemoglobin content rose after this handling from an average of. mg. per 00 ml. to. mg. per 00 ml., and from a minimum of 0 to a maximum of. mg. If the ratio of the amount of supernatant plasma to packed cells is roughly to., it can be seen that in these bloods before agitation about onequarter of the free hemoglobin is in the supernatant plasma, and three-quarters is trapped with the packed cells. This is, however, quite variable. Sample No. showed no increase, whereas in sample No. 0, only one twenty-third of the hemoglobin found in the specimen was in the supernatant plasma. It is safe to conclude that the diffusion of hemoglobin through a specimen of banked blood varies widely and that any estimate of the free hemoglobin content based on what is seen in the supernatant plasma alone is likely to. be grossly inaccurate. Downloaded from on 0 January 0

3 GROVE-RASMUSSEN AND SOUTTER TABLE AMOUNT OF FREE HEMOGLOBIN IN MG. PER 00 ML. IN THE SUPERNATANT PLASMA OF EACH OF 0 BOTTLES OF BANKED, ARRANGED ACCORDING TO SAMPLE N'O. S 0 o..s. S'.O S.O...S.S..... S... IS 0 ' S S 0 S 0 s s s s IS.0 S.O S.O TABLE AMOUNT OF FREE HEMOGLOBIN IN MG. PER 00 ML. IN EACH OF 0 SPECIMENS OF BANKED AFTER RESUSPENSION OF THE CELLS, ARRANGED ACCORDING TO S S.O S Table gives a comparison of results of two methods of removal of the red cells in the first 0 specimens after resuspension. In the first column (A) are Downloaded from on 0 January 0

4 HEMOGLOBIN IN PLASMA AND SERUM listed the hemoglobin contents of the plasma after centrifuging by the method described in the preceding paragraph; in the second column (B) are given the hemoglobin contents of the plasma after allowing the cells to settle out for six hours in a refrigerator. In both methods the plasma was aspirated, centrifuged, re-aspirated and tested. There was a maximum difference between the methods of. mg. and a minimum of 0. The average was 0. mg. These figures are within the per cent error of the method of measurement of the hemoglobin content, and therefore have no significance. On the basis of the consistent findings in this small group, it was felt that the centrifuging method introduced no appreciable error and could well be used throughout the rest of the study. The original 0 bloods and an additional 0 were filtered in the usual manner, blood aspirated after this process was then centrifuged, the plasma aspirated, re-centrifuged and re-aspirated. TABLE A COMPARISON OF THE FREE HEMOGLOBIN IN WHOLE BANKED WHEN THE PLASMA IS OBTAINED BY CENTRIFUGING (COLUMN A) OR BY GRAVITY (COLUMN B) A HEMOGLOBIN IS MG./00 ML. B 0.0. S S The amounts of free hemoglobin measured in these samples are listed in Table. They give a good idea of the amount to be expected in blood as it leaves our bank. The minimum amount of free hemoglobin was. mg. per 00 ml., the maximum., the average.0 mg. If the average donor has grams of hemoglobin per 00 ml. before his blood is diluted with ml. of citrate, his blood will have Gm. afterwards. On this basis, for a flask of 00 ml. the average amount of liberated hemoglobin would then be 0. per cent, the maximum 0. per cent. The ages of the blood studied are typical of those bloods given out by the bank; per cent had been stored seven days or less, per cent from one to two weeks and per cent from two to three weeks. In the first 0 bloods which were aspirated before filtering and again afterwards, there was a definite rise of the hemoglobin content after filtration. The minimum rise was 0, the maximum. and the average. mg. or about per cent. This rise can possibly be accounted for by (a) a better mixture of the hemoglobin trapped among the cells with the plasma, (b) further liberation of hemoglobin due to some cell destruc- Downloaded from on 0 January 0

5 GROVE-RASMUSSEN AND SOUTTER tion by the filtration process, (c) slight hemolysis caused by moistening the filter with saline and (d) hemolysis caused by warming the blood through con- TABLE. AMOUNT OF FREE HEMOGLOBIN IN MO. PER 00 ML. IN 0 SPECIMENS OF WHOLE BANKED WHEN DISPENSED, ARRANGED ACCORDING TO SAMPLE NO. MG. OF. HB. SAMPLE NO. MG. OF HB. SAMPLE NO. MG. OF HB. SAMPLE NO. MG. OF HB.... S S...0 / IS...0.S S S S S S TABLE HEMOGLOBIN CONTENT ACCORDING TO AGE PINTS OF DISPENSED PER CENT HEMOGLOBIN IN PLASMA MG./0Q ML. Lowest Value Highest Value Average Value -..S tact with the filter, funnel and fresh blood-bottle, all of which are stored at room temperature. In Table, the hemoglobin contents of the 00 samples of filtered blood are grouped according to the age of the specimens. The group taken from the third Downloaded from on 0 January 0

6 HEMOGLOBIN IN PLASMA AND SERUM week is unfortunately small, but the average values show the expected upward trend associated with length of storage that so many writers have previously noted. During the period when these studies.were being carried out, our pyrogenic reaction rate for transfusions rose from to per cent. This was attributable to improperly cleaned tubing; a change in the method of the preparation of the tubing led to a reduction of the reactions to about. per cent a short time later. Associated with the administration of these 00 bloods, there were reactions on the part of the recipients. The reactions were typically pyrogenic, characterized by chills and fever, and without hemoglobinuria. In each instance the groupings, Rh factor determinations and cross matchings were checked and found to be correct. In Table G the relationship between these reactions and the amount of free hemoglobin in the transfusions received is shown. It can readily be seen that the reactions, although small in number, have a statistical relationship to TABLE REACTIONS IN RELATION TO HEMOGLOBIN CONTENT FREE HEMOGLOBIN PINTS OF PER CENT REACTION'S mg./loo ml S S the number of bloods dispensed and are not proportional to the amount of free hemoglobin. II. HEMOGLOBIN LEVEL OP SERUM OF PATIENTS DURING FEBRILE FOLLOWING TRANSFUSION REACTIONS Flink has shown that transfusion of whole blood unattended by reactions is unaccompanied by hemoglobinuria. He and other investigators have stressed the importance of examining the serum of patients immediately following febrile reactions to distinguish between pyrogenic and true hemolytic reactions. Chills and fever almost always accompany pyrogenic reactions. They are, however, inconsistent companions of hemolytic reactions and usually are absent in urticarial and other allergic reactions. In cases of gross hemoglobinuria following administration of mismatched blood, we have observed two patients who gave no evidence of any reaction during or following transfusion. If the liberation of hemoglobin in large quantities does not always cause fever, does it follow that fever during or following transfusions will consistently be accompanied by some measurable increase in the free hemoglobin content of the recipient's serum? Under usual conditions, there is in the serum of a healthy person a detectable amount of free hemoglobin. As a control in this series, blood was taken from persons and studied to ascertain the average hemoglobin level. Downloaded from on 0 January 0

7 0 GKOVE-EASMUSSEN AND SOUTTER METHOD Twenty-five blood donors were selected, their histories were taken, they were physically examined and their temperature, blood pressure and hemoglobin levels were determined. In each of them, the hemoglobin level was at least. Gm. per 00 ml. blood. The blood to be tested was taken following withdrawal of 00 ml., by cutting the tubing and allowing the blood to flow into a bottle. The negative pressure created by withdrawing blood with a dry syringe and needle will often cause a small amount of hemolysis, enough to interfere with the calculations in a series of this sort. If two different specimens are obtained by the syringe method at the same time, and both have the same free hemoglobin content, the probability that the hemolysis occurred intravascularly is greatly increased. For purposes of this series, it was easier to eliminate this source of error by eliminating the use of the syringe entirely. The bloods obtained were taken into clean, dry test tubes containing no anti-coagulant. They were placed in a refrigerator for from one to two hours to permit clot formation. No mechanical method of loosening the clot (as with a wooden applicator stick) was employed for fear of causing some hemolysis. Instead, they were centrifuged for five minutes at 00 r.p.m. The supernatant serum was then aspirated and again centrifuged at the same speed for a similar length of time. The supernatant serum was again aspirated and the amount of free hemoglobin was measured by the method previously described. The free hemoglobin level in the serum of the normal persons varied from. to. mg. per 00 ml. Having established a rough base line for the hemoglobin content of the serum of normal persons, a search was made for patients with typical pyrogenic reactions, the criteria for which were: first, that the patient should have no disease that might give him fever, hemolysis, or chills; second, that no medication or treatment had been administered during or before transfusion, which was capable of producing hemolysis; third, that the symptoms, namely chills and fever, be present in unmistakable form. A fresh sample of his blood was to be taken after the reaction along with some blood from the transfusion bottle for re-grouping, re-testing for the Rh factor and re-crossmatching. The blood samples to be tested were taken as soon after the onset of symptoms as possible, certainly before three hours had elapsed. As various writers have pointed out, during a hemolytic reaction the hemoglobin will appear in the blood serum immediately after the appearance of the first symptoms, and will gradually disappear after a few hours. 0, We have verification of these reports from a hemolytic reaction which we observed: An Rh-negative Group O patient, after receiving ml. of Rh negative, Group A blood developed a violent reaction characterized by chills, epigastric and lumbar pains, and flushing of the skin, so that the infusion was stopped and the error detected. A specimen of blood was withdrawn immediately from the patient, unfortunately by means of a syringe. It was centrifuged and the adherent clot loosened with a small stick, so that no accurate measurement of the free hemoglobin could be made because of the possibility of artificially-induced hemolysis. A second specimen, withdrawn one hour and forty minutes after the reaction, showed a free pigment level of mg. per Downloaded from on 0 January 0

8 HEMOGLOBIN IN PLASMA AND SERUM 00 ml. A third specimen, four and one-half hours after the onset of the reaction, showed. mg., and a fourth specimen, sixteen and one-half hours from the onset of symptoms, showed. mg. It is difficult, unfortunately, to obtain immediate notification of pyrogenic reactions from the wards; hence, the time of collection of the first specimen after the onset of the reaction is not uniform. Furthermore, the first five speci- TABLE AMOUNT OF FREE HEMOGLOBIN IN MG. PER 00 ML. IN PATIENTS WITH FEBRILE TRANSFUSION REACTIONS SPECIMEN MG. FREE HEMOGLOBIN IN SERUM PER 00 ML. IN RELATION TO TIME AFTE*R ONSET OP THE TRANSFUSION REACTION min. min. 0 min. lhr. l'ahr. hr.' 'Ahr. hr ' S ' mens collected were withdrawn through a -gauge needle by means of a single, dry syringe. Three of these specimens (nos., and ) showed values higher than normal levels, which may be due to hemolysis resulting from negative pressure in the syringe. The last 0 specimens were taken by inserting an - gauge needle into the patient's vein and allowing the blood to flow into a test tube. Blood samples were examined in the same manner as were those from the normal donors. The results are listed in the appended table. As will readily be noticed, with the exception of specimens, and, the amount of detectable free hemoglobin was about the same as for the controls, the levels ranging from. to.0 mg. per 00 ml. Downloaded from on 0 January 0

9 GROVE-RASMUSSEN AND SOUTTER SUMMARY AND CONCLUSIONS A study of the free hemoglobin content in 00 specimens of banked blood is presented. The amount of free hemoglobin in the supernatant plasma of banked * blood bears an extremely variable relationship to that in the blood as a whole: on an average about only one-quarter of the total free hemoglobin is in the plasma. This observation renders highly inaccurate any estimate of the amount of free hemoglobin contained in a blood that is based on observing the supernatant plasma. Filtering these banked bloods increased the hemoglobin content by about. mg. per 00 ml. The average content of free hemoglobin in the 00 bloods studied was.0 mg. per 00 ml., the maximum. mg. The amount of hemoglobin increased with length of storage, but bore no quantitative relationship to febrile reactions on the part of the recipients. The largest amount of free hemoglobin found in any of the specimens was but a sixteenth of the amount described by some authors as safe to give to a patient. Specimens of blood from patients who had had typical febrile (pyrogenic) reactions were studied to determine the amount of hemoglobin which had been liberated. In those cases in which the blood had been taken without artificially induced hemolysis, there was no rise above the normal levels found in controls, but the levels were all much lower than those found in a patient who had had a typical hemolytic reaction after receiving approximately ml. of mismatched blood. This series gives evidence that hemolysis does not follow-pyrogenic reactions and, conversely, that if hemolysis is present following a reaction, one should suspect a transfusion of mis-matched blood, provided other factors causing hemolysis can be excluded. REFERENCES. CARLSON, R. I.: Reactions to transfusions of banked blood. Yale J. Biol, and Med., : -,.. FAIRLEY, N. Y.: The fate of extracorpuscular circulating hemoglobin. Brit. M..]., : -, 0.. FLINK, E. B.: Blood transfusion studies. I. The differentiation of hemolytic and nonhemolytic transfusion reactions. J. Lab. and Clin. Med., : 0-0,.. GILLIGAN, D. R., ALTSCHULE, M. D., AND KATERSLEY, E. M.: Studies of hemoglobinemia and hemoglobinuria produced in man by intravenous injection of hemoglobin solutions. J. Clin. Investigation, 0: -,.. HUNTER, F. T.: Personal communication to the authors.. HUNTER, F.'T., GROVE-RASMUSSEN, M., AND SOUTTER, L.: A spectrophotometry method for quantitating hemoglobin in plasma or serum. Am. J. Clin. Path., 0: -, 0.. MOLLISON, P. L.: The investigation of hemolytic transfusion reactions. Brit. M. J., : -,. S. MUETHER,.R. O., AND ANDREWS, K. R.: Studies on stored blood. II. Effect of storage on human blood. Am. J. Clin. Path., : -0,.. MUETHER, R. O., AND ANDREWS, K. R.: Studies and uses of stored blood and plasma. South. M. J., : -,. 0. OTHLECKEH, F.: Die Blut. Transfusion. Handbuch Allg., Vol., Part. Berlin: Hamat,, pp. -.. SOUTTER, L.: Procedures of the blood bank at the Massachusetts General Hospital. New England J. Med., 0: -,.. WIENER, A. S.: Blood Groups and Transfusion. Ed.. Springfield,.: Charles C Thomas,, pp. -.. WIENER, A. S., OREMLAND, B. H., HYMAN, M. A., AND SARNWICK, A. A.: Transfusion reactions. Am. J. Clin. Path., : 0-,. Downloaded from on 0 January 0