EFFECT OF HEATING ON THE OSMOTIC FRAGILITY OF STORED BLOOD

Transcription

1 Br.J. Anaesth. (1978) 50, 815 EFFECT OF HEATING ON THE OSMOTIC FRAGILITY OF STORED BLOOD J. H. VAN DER WALT AND W. J. RUSSELL SUMMARY The effect of incubation at 37 C, 46 C, 47 C and C for 1 h on the osmotic fragility of blood was determined. Blood taken from volunteers into ACD and CPD was examined after 1, 7, 14 and 21 days of storage. In addition, 22-day-old blood stored in CPD was examined. The osmotic fragility was unchanged after incubation at 37 C and 46 C C, slightly increased at 47 C and markedly increased at C. The duration of storage did not have any marked effect. The results suggest that incubation of stored blood at 46 C for 1 h does not cause red cell damage. The rapid infusion of large amounts of blood can be life-saving. However, the rapid infusion of cold blood produces a significant and possibly dangerous decrease in body temperature. Since the direct cooling of the heart may provoke cardiac arrhythmia and even arrest, it is customary to warm stored blood before large transfusions. Ideally, the blood should be heated to body temperature (37 C), but temperatures greater than 32 C are acceptable (Russell, 1974). Most commercially available systems have operating temperatures between 37 and 39 C. With a polyvinyl chloride coil, the output temperature is 30 C or less at a flow rate of 150 ml min" 1 in a bath at 37 C (Russell, 1974). If the bath temperature could be increased by several degrees without damage to the red cells, blood could be delivered to the patient at a higher temperature and the danger of a cool infusion during rapid replacement would be avoided. Chalmers and Russell (1974) showed that frank haemolysis is evident at 50 C but not at 45 C or less. However, it was possible that less obvious damage was occurring at 45 C and less, damage that would still render the red cells unsuitable for oxygen transport. Such a change might be caused by a denaturation of the cell protein. J. H. VAN DER WALT,* M.B., CH.B.(CAPE TOWN), F.F.A.R.C.S. (IRE.); W. J. RussELL,t M.B., B.S.(ADELAIDE), F.F.A.R.C.S., F.F.A.R.A.C.S., PH.D., D.I.C.J Department of Anaesthesia, Royal Postgraduate Medical School, DuCane Road, London W12 OHS. Present addresses: * Department of Anaesthesia, Red Cross War Memorial Children's Hospital, Cape Town, South Africa. t Department of Anaesthesia and Intensive Care, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia, /78/ $01.00 One indication of subtle damage might be impairment of the ability of the red cell membrane to resist osmotic strain. Thus a series of experiments on osmotic fragility using ACD and CPD blood of various ages from 1 to 22 days was undertaken. In addition, these experiments gave us the opportunity to define more precisely the temperature at which damage occurred. METHODS Material (a) Twenty-two-day-old CPD blood was selected from the hospital blood bank. Reproducibility estimates were made on this blood, and measurements of osmotic fragility after incubation. (b) Blood was collected into Fenwal donor packs containing either ACD or CPD anticoagulants from six healthy male adults ranging in age from 30 to 40 yr. The appropriate amount by weight of anticoagulant was removed from each pack to provide the correct ratio of anticoagulant for 250 ml of blood. This was achieved by subtracting the weight of a completely empty pack from the pack with anticoagulant. The proportion by weight of anticoagulant was calculated for 250 ml of blood and the rest of the anticoagulant removed. Two hundred and fifty millilitre of blood was collected by venepuncture in the cubital fossa into ACD or CPD and then, by inserting the collection needle of the other pack into the rubber part of the collecting tube, a further 250 ml was withdrawn similarly into the alternative anticoagulant. Thus, a single donation of 500 ml of blood was distributed equally between ACD and CPD anticoagulants. Adequate mixing during collection was achieved by Macmillan Journals Ltd 1978

2 816 BRITISH JOURNAL OF ANAESTHESIA continual gentle agitation of the packs. After collection, the blood was stored at 4 C in a blood refrigerator. The haematocrit values from ACD and CPD packs were measured to check the dilution; the values for one subject did not differ by more than 1.4%. Heating At 1 day, 1 week, 2 weeks and 3 weeks, four samples were taken and heated to 37 C, 46 C, 47 C and C. On removal from the refrigerator the blood was gently agitated for 5 min. The blood samples were agitated also for 1 min to ensure a uniform haematocrit before sampling. The blood was returned to the refrigerator immediately after sampling. Samples were taken by free flow into clean dry 10-ml centrifuge tubes. Each sample nearly filled the tube, and care was taken to avoid a film of blood on the side of the tube above the meniscus. The tube was then stoppered and incubated at a specific temperature: 37 C, 46 C, 47 C or C for 60 min in a Gallenkamp Shaking Reaction Incubator. Osmotic fragility Buffered saline (NaCl-PO 4 ) solutions of varying concentrations were selected (Parpart et al., 1947). The concentrations used were 0.85, 0.70, 0.65, 0.60, 0.55, 0.50, 0.475, 0.45, 0.40, 0.35, 0.15 and 0.10%. Complementary solutions to restore the saline to isoosmotic concentration were omitted as by Dacie and Lewis (1968) in their modification of the method. For each incubated blood sample, a set of 12 centrifuge tubes was made up to have 5 ml of each concentration of buffered saline. After incubation, the blood tube was inverted twice. A 0.02-ml sample of heated blood was transferred to each tube of the set. Each tube was inverted once and then allowed to stand for 30 min at a temperature of C. After standing, the tubes were centrifuged at 1500 rev min- 1 for 5 min. The supernatant was decanted carefully and its optical density read directly in a colorimeter (Las Ljungberg, Sweden) through a 540-nm filter with the zero set on a distilled water blank. The optical density of the 0.85% saline was subtracted from all readings. The mean optical density change with the 0.1% saline was taken as 100% haemolysis. Sources of error All volume measurement was estimated to be ± 2.5% of the nominal value. A more serious potential source of error was haemolysis caused by the anticoagulant, trauma, drying of blood in the tubes or water condensation during incubation. However, the 0.85% saline reading was never more than 2% of the optical density of full haemolysis after incubation at 47 C and less in the blood from volunteers. The reproducibility of the results was checked in two ways. Duplicate samples were run on the 0.1% saline for 265 estimates. The mean difference of the optical density was 2.14% (SD 1.69). A further overall estimate was made on six samples from a single unit of 22-day-old CPD blood. The coefficient of variation for the six estimates of the saline concentration for 50% haemolysis (H 50 ) was 0.%, the coefficient of variation for the slope of the haemolysis line was 3%. Analysis of results Parpart and others (1947) presented the osmotic fragility curve on probability paper using the central values. A similar procedure was followed in this study. The saline causing 50% haemolysis (H 50 ) and rate of haemolysis was estimated from 10 to 95% haemolysis between 0.6% and 0.4% saline. Usually, six values were used; occasionally, particularly at C, fewer values met the criteria. At least three values were always used and the values always HAEMOLYSIS (%> SALINE (%) A A J, FIG. 1. Osmotic fragility curves for blood incubated at 37 C (circles) and C (triangles) on a linear probability plot. The lines are the best fit probability lines using the points encompassed by the lines. At 37 C H 50 was and sigma was Thus 15.8% of cells, one standard deviation above the mean or H 50J haemolysed at 0.584% saline (0.527 ±0.0565) and 84.1% of cells, one standard deviation below the mean or H 50, haemolysed at 0.470% saline. Similarly, at C H 50 was and sigma was , thus 84.1% haemolysis occurred at 0.549% saline. The 15.8% haemolysis is not shown.

3 OSMOTIC FRAGILITY OF STORED BLOOD AFTER HEATING 817 bracketed the H B0 ; occasionally the 0.65% value was included for this. The H 50 and rate of haemolysis were estimated by means of the BMD Probit Analysis program using a l-x transform (Dixon, 1976). The H 50 is the saline concentration which causes sufficient osmotic stress to haemolyse 50% of the red cells. The rate of haemolysis is expressed by sigma. This is the change in saline concentration necessary to increase haemolysis of the red cell population by one standard deviation, that is to increase haemolysis from 15.86% to 50%, from 50% to 84.13% or 84.13% to 97.72% of the population. Sigma is the reciprocal of the probit slope (fig- 1). Where possible, all analyses were performed using a paired t test. All P values given are for a two-tailed result. RESULTS The incubation of anticoagulated blood for 1 h did not alter greatly the osmotic fragility of the blood until the incubation temperature reached C, when slight haemolysis was present. 22-day-old CPD blood Samples were taken from six packs of blood and incubated at one of the selected temperatures. The haemolysis lines for blood from the same pack H Incubation Temperature CO FIG. 2. The change in saline concentration causing 50% haemolysis (H 50 ) with incubation temperature for 22-dayold bank blood. H 50 was greater at C, indicating an increased red cell susceptibility to disruption by osmotic stress. Each symbol is the mean of six estimates. The stem and bar indicates 1 SD. Values represented on the graph (temperature ( C); H 50 (mean±sd)) and the SD of change from 37 C are: ± ± The change in H 60 was significantly different from 37 C at 47 C (f = 8.11, P < 0.001) and at C (r = 18.7, P < 0.001). incubated at 37 C and at C are shown in figure 1. The fragility curve for the blood incubated at C is shifted to the left, that is haemolysis occurs at greater concentrations of saline, and slope of the curve is less. Thus the cells are more fragile and begin to disrupt at lower osmotic gradients. However, the amount of further disruption caused by each osmotic step is smaller. The changes in H 50 with incubation temperature are shown in figure 2, and the changes in sigma in table I. A small but statistically significant change in H 50 and the rate of haemolysis occurred at 47 C. At C H 50 was much greater and the curve was shifted markedly to the left. In addition there was a considerable decline in the rate of haemolysis. TABLE I. The haemolysis slope changed with incubation temperature for 22-day-old CPD bank blood. Significance levels refer to a paired t test with the same blood at 37 C. Temp Sigma mean SD = < Blood from volunteers Haemolysis with heating. Most of the samples heated to C showed some haemolysis; this was apparent as an increase in the optical density for the 0.85% buffered saline above that for the 37-, 46- and 47- C samples. Haemolysis was present in both the ACD and CPD blood samples. The mean increase in optical density for all samples at C corresponded to 2.2% haemolysis. CPD versus ACD. The blood from six volunteers was collected in both ACD and CPD. Blood with each type of anticoagulant showed a sharp increase in osmotic fragility at C. However, at all incubation temperatures H 50 for ACD blood was at a lower osmotic stress (higher concentration of saline) than that for CPD blood (fig. 3). Although the haemolysis TABLE II. The haemolysis slope changed with incubation temperature (mean ± SD) in volunteer ACD and CPD blood. Significance level refers to a paired t test Temp. ( C) ACD CPD P ± = ± ± ± ± ±0.0114

4 818 BRITISH JOURNAL OF ANAESTHESIA Incubation Temperature ( C) FIG. 3. The effect of incubation temperature on the H 50 of volunteer ACD and CPD blood. Each point is the mean of 24 estimate blood samples. The stem and bar indicate 1 SD. At all incubation temperatures, H 50 for ACD blood was at a lower osmotic stress than that for CPD blood: Concentration of saline producing 50% haemolysis (,H 50 ) in ACD and CPD volunteer blood after 1 h incubation at various temperatures {mean + SD), SD of difference and P value by a paired t test are given over 24 samples CPD ACD SD dlff ± ± < ± ± ± < ± ± ± < ± ± ± < rate tended to be less for ACD blood, only the slope value for samples incubated at 37 C was statistically significant by a paired test (table II). Age of blood. Ageing affected H 50 in different ways depending on the anticoagulant. There was no marked trend in the rate of haemolysis with either anticoagulant. In ACD blood, H 50 after incubation at all temperatures was unchanged for the first 14 days of storage. However, there was a decreased fragility at 21 days (table III). In CPD blood, H 50 after incubation at all temperatures increased progressively, indicating an increasing fragility with age (table IV). At 21 days the fragility as measured by H 50 for each incubation temperature was similar for ACD and CPD blood. When H 50 at each of the incubation temperatures was examined at each age ACD blood did not alter greatly over 21 days. Paired statistical comparison showed a significantly greater H 50 saline at 47 C than at 37 C in only the 1-day samples. The difference was very small and less than the difference in H 50 between subjects at 37 C incubation. With CPD blood, paired comparison suggested that some change with age may have occurred. There was a significantly greater H 50 at 47 C than at 37 C with the 14- and 21-day samples; however, again this difference was small. TABLE III. The effect of storage time on H so after incubation at various temperatures of ACD blood. The mean values of six samples are shown with the standard deviations. P values are for 1 day compared with 21 days storage Temp. - ( Q ± ±0.021** ±0.028* ± ±0.014** Day ± ± ±0.028** ± ± * Significantly different from the same day samples incubated at 37 C: *P<0.05; **P<0.01. P = 0.04 = = TABLE IV. The effect of storage time on // 60 of CPD blood after incubation at various temperatures. The mean values for six samples are shown with the standard deviations. The P values are for 1 day compared with 21 days storage Temp. - ( C) 1 7 Day P ± ± ± ±0.018*** ± ±0.020** *** ± ±0.021* ±0.033** Significantly different from same day samples incubated at 37 C: *P<0.05; **P<0.01; ***P< < = 0.002

5 OSMOTIC FRAGILITY OF STORED BLOOD AFTER HEATING 819 DISCUSSION This work complements the previous work of Chalmers and Russell (1974). They showed that incubation at 45 C had no haemolytic effect, but incubation at 50 C did. This corresponds with the observation in the present study that the optical density of the isotonic saline increased in samples incubated at C, presumably because a small amount of haemolysis was occurring. Estimates of the osmotic fragility measure the ability of the red cells to resist disruption. An increase in haemolysis could be caused by a loss of membrane flexibility or a change in shape to a less compliant form. Quite possibly, heat could have both effects, denaturing the membrane and increasing red cell volume. An explanation of the consistently better resistance to osmotic stress by the CPD-stored blood (fig. 3) could be that CPD dehydrated the red cells so that their initial cell volume was less. However, this was not supported by the haematocrit values, which were similar for both ACD and CPD stored blood. An alternative explanation is that CPD storage was better able to sustain the red cell metabolism and hence the integrity of the cell membrane. The osmotic fragility curve appeared to be a sensitive index of heat damage. There was a marked shift in H 50 at C and a mild but significant shift at 47 C. However, without the precise comparison possible with the same blood treated at each temperature, the slight shift to the left in H 50 for 47 C would have been masked by the H 50 population variation. The change in the haemolysis rate is of interest as it suggests that heat damage was not occurring equally throughout the population, but was concentrated in the cells already susceptible to damage by osmotic stress. Possibly, heat damage caused more denaturation in cell membranes already showing a loss of elasticity, or there was an acceleration of the degeneration of some process such as the ionic selectivity in the cell membrane. It seems possible that the haemolysis rate might prove a more sensitive indicator of abnormal cell populations than H 50. The ACD anticoagulant appeared to cause an increase of red cell fragility very early during storage but this was not progressive. The slight decrease in fragility at 3 weeks is puzzling, but occurred in the ACD blood from five of the six volunteers. With CPD anticoagulant there was a progressive increase in fragility, but initially the red cells were less fragile than those stored in ACD and equal osmotic fragility did not occur until 3 weeks storage. The difference between CPD bank blood and CPD volunteer blood was marked. At all incubation temperatures the H 50 and sigma values were greater for bank blood. This related presumably to the conditions of collection and storage because there was only 1 day difference in age between the oldest volunteer blood and bank blood. Certainly the volunteer blood was collected and kept under optimal conditions. There was continuous agitation during collection and the blood was maintained carefully at 4 C during storage. The bank blood incubated at 37 C had a greater H 50 and sigma than the 21-day volunteer blood incubated to 47 C. Possibly, collection and storage conditions can affect blood more adversely than a moderately severe exposure to heat for 1 h. The small changes in fragility at 47 C were probably indicative of the beginning of damage to the red cell membrane. It is known that haemolysis at 50 C for both ACD and CPD blood increases progressively with the time of incubation (Chalmers and Russell, 1974). Possibly, a more prolonged incubation may have produced a more marked effect. It would seem that red cell damage begins at 47 C and is obvious at C, after 1 h of incubation. There was no evidence of damage with incubation at 46 C. It would seem reasonable that a blood warmer could have its safety cut-out set to a maximum of 46 C when in use with massive transfusion. Blood is unlikely to remain in the system for a period exceeding 1 h, so this study covers the likely duration of incubation in clinical practice. A regulating thermostat set to 44 C would give good warming even at fast flows and have a 2- C margin from the safety cut-out. ACKNOWLEDGEMENTS We would like to thank our colleagues in the Department of Anaesthesia at the Royal Postgraduate Medical School, London, for their advice and criticism, and the Blood Transfusion Service for their co-operation during this study. During the preparation of this article, Dr W. J. Russell was supported by the Wellcome Trust. We also thank Miss E. Hilton for secretarial assistance. REFERENCES Chalmers, C, and Russell, W. J. (1974). When does blood haemolyse? Br.J. Anaesth., 46, 742. Dacie, J. V., and Lewis, S. M. (1968). Practical Haematology, 4th edn, p London: Churchill. Dixon, W. J. (1976) (Editor). BMD Biomedical Computer Programs. BMD03S Biological Assay: Probit analysis., p Berkeley: University of California Press.

6 820 BRITISH JOURNAL OF ANAESTHESIA Parpart, A. K., Lorenz, P. B. 5 Parpart, E. R., Gregg, J. R., and Chase, A. M. (1947). The osmotic resistance (fragility) of human red cells. J. Clin. Invest., 26, 636. Russell, W. J. (1974). A review of blood warmers for massive transfusion. Anaestk. Intent. Care, 2, 109. EFFET DU CHAUFFAGE SUR LA RESISTANCE GLOBULAIRE DU SANG CONSERVE RESUME On a determine l'effet de l'incubation a 37, 46, 47 et C sur la resistance globulaire du sang. Le sang, qui avait ete preleve sur des volontaires et place en ACD et en CPD, a ete examine apres une periode d'entreposage de 1, 7, 14 et 21 jours. On a, en outre, examine du sang entrepose en CPD pendant 22 jours. La resistance globulaire est demeuree sans changement apres incubation a 37 C et a 46 C, mais elle a legerement augmente a 47 C et fortement augmente a C. La duree de l'entreposage n'a pas produit d'effet marque. Les resultats obtenus laissent penser que l'incubation du sang entrepose a 46 C durant 1 h n'endommage nullement les globules rouges. AUSWIRKUNG DER ERWARMUNG AUF DIE OSMOTISCHE HINFALLIGKEIT GESPEICHERTEN BLUTES ZUSAMMENFASSUNG Die Auswirkung der Inkubation bei 37, 46, 47 und C in einer Stunde auf die osmotische Hinfalligkeit von Blut wurde bestimmt. In ACD und CPD aufgenommenes Blut von Freiwilligen wurde nach Speicherungsdauer von 1, 7, 14 und 21 Tagen untersucht. Zusatzlich wurde Blut untersucht, das 22 Tage in CPD gespeichert war. Die osmotische Hinfalligkeit war nach Inkubation bei 37 und 46 C unverandert, leicht erhoht bei 47 C und stark erhoht bei C. Die Dauer der Speicherung hatte keine erkennbare Auswirkung. Diese Ergebnisse zeigen, dass die Inkubation von gespeichertem Blut fiir 1 Stunde bei 46 C keine Schadigung der roten Blutkorper bewirkte. EFECTO DE CALENTAMIENTO SOBRE LA FRAGILIDAD OSMOTICA DE SANGRE ALMACENADA SUMARIO Se determino el efecto de incubaci6n a 37, 46, 47 y C durante 1 h sobre la fragilidad osmotica de la sangre. Se examino la sangre tomada de voluntarios y colocada en ACD y CPD al cabo de 1,7,14 y 21 dias de almacenamiento. Ademas, se examino la sangre almacenada en CPD al cabo de 22 dias. La fragilidad osmotica no acuso cambios al cabo de incubacion a 37 y 46 C, pero aumento ligeramente a 47 C y aumento notablemente a C. La duration del almacenamiento no ejercio efecto notable alguno. Los resultados sugieren que la incubacion de la sangre almacenada a 46 C durante 1 h no causa dano a los globulos rojos.