- Email: [email protected]
TREATMENT OF RESPIRATORY-DISTRESS SYNDROME
281
finding of paramyxovirus-like nucleocapsids in the tissueculture fluid (fig. 2). Fig. 3 shows a measles virus for comparison. The cytopathic agent was ether sensitive and resistant to 5-bromo-3-deoxy-uridine D.N.A. Although the finding of a paramyxovirus-like agent is of great interest it must be interpreted with considerable caution in view of well-recognised contamination dangers, especially when measles is in progress simultaneously. However, it is hoped that this brief report together with that of Dr. ter Meulen and his colleagues will encourage further efforts
at
virus isolation with newer methods. E. J. FIELD S. COWSHALL H. K. NARANG T. M. BELL.
M.R.C. Demyelinating Diseases Unit, Newcastle General Hospital,
Westgate Road, Newcastle upon Tyne NE4 6BE.
PLASMA AND ERYTHROCYTE MAGNESIUM LEVELS IN AFFECTIVE DISORDERS
SIR,-We should like to comment on the controversy magnesium levels in depression. Herzberg and Bold1 reported a significant difference in serum-magnesium levels between male and female depressed patients, but Carney and Sheffield2 could not confirm these results. Cade3 found raised serum levels during and after depression, whereas Frizel et awl. found the opposite-viz., serum-levels were low during depression and increased on recovery. Bjorumfound no difference between depressed and recovered patients, but the mean value in patients was higher than in controls. During a double-blind cross-over trial s of prophylactic lithium in affective disorders, samples of plasma and erythrocytes were analysed for magnesium, sodium, potassium, and lithium ions, using an atomic-absorption spectrophotometer (’ SP 90’, Pye Unicam). During the 6-month placebo period, samples (on average, three per patient) were taken every 1-2 months, from six male and nine female outpatients (the majority bipolar manic depressives). Preliminary analysis of our data reveals a significant difference in erythrocyte magnesium levels (expressed as mean±s.D. with number of observations in parentheses) between males, 4-77:0-45 meq. per litre (16), and females, 4-54:0-60 (25) (analysis of variance; P < 0-05). The mean plasma-magnesium in males, 1-890-21 (16), also seemed to be greater than in females, 1-82:0-15 (24), but this difference was not statistically significant. There were no significant differences in plasma or erythrocyte levels of sodium or potassium between males and females. Samples taken (on average, four per patient) from the same patients during 6 months on lithium carbonate showed no significant difference in lithium levels, in plasma or erythrocytes, between males and females. Lithium did not produce any significant alterations in plasma or erythrocyte levels of magnesium, sodium, or potassium. In view of the central role of magnesium in energyproducing enzyme reactionsthere is a need for further study of magnesium metabolism in affective disorders, with special reference to age and sex differences. over
One of
us
(R.
L.
C.)
was
supported by
a
Wellcome research
SALMONELLOSIS IN LIVESTOCK illusion about the ample evidence in the spread of salmonellosis are only too frequently reminded by the Public Health Laboratory Service of the human involvement in outbreaks of salmonella food-poisoning which regularly occur after wedding receptions and other such functions. I am sure both veterinary and medical personnel involved with the epidemiology of salmonellosis are fully aware of the animal origin of the causal organism and would totally disagree with Dr. Anderson’s playing-down of the human element in the spread of the disease. The investigation and control of any disease requires short-term immediate action which may be dispensed with once suitable long-term measures become available. As we know that salmonellas enter food establishments on some contaminated meat and poultry, is it too much to ask that the organisms be prevented from proliferating or spreading to wholesome food because of infected humans and poor hygienic control of food-handlers ? Would this be " wasted effort ", especially since salmonellosis in our animal population is not going to be controlled overnight.
SIR,-Ihave
no
incriminating humans (July 15, p. 138). We
University of Liverpool Department of Veterinary Medicine, Field Station, Leahurst, Neston,
JOHN
Wirral, Cheshire L64 7TE.
R. WALTON.
TREATMENT OF RESPIRATORY-DISTRESS SYNDROME
SIR,-Recent articles and correspondence in
your
journal suggest that the treatment of respiratory-distress syndrome of the newborn (R.D.S.) should be approached from a new angle. In the past, no treatment has been started until symptoms have developed, but new findings 1-3 suggest that the development of R.D.S. can be predicted at birth or even before. Therefore, if R.D.s. can be predicted, should start at birth to try to prevent the onset of symptoms, since it is generally accepted that R.D.s. is a self-limiting condition,4provided the child can be kept alive. The molecular oxygen in the inspired air may be toxic 5-6 and a raised " inspired " Po2 has many other dangers,7,8 but the arterial Po2 can be maintained with a lower inspired Po2 by continuous positive pressure applied to the airway,9 and use of this technique with an inspired Po2 of less than the 20% in normal air may be beneficial. Atelectasis and airway closure is one factor in the development of hypoxia 2,9 and, by substituting helium for nitrogen in the inspired gases, laminar flow may be preserved in some of the very small air passages, thus preventing atelectasis. Once hypoxia has developed, a vicious circle may be set up with the hypoxia causing pulmonary arteriolar constriction, resulting in further hypoxia.Halothane can relax the pulmonary circulation 10 and, by adding halothane to the inspired gases, a sufficient concentration may be achieved in the pulmonary vascular tree to counteract the hypoxic
treatment
fellowship in clinical pharmacolosv. M.R.C. Brain Metabolism Unit, University Department of Pharmacology, 1 George Square, Edinburgh EH8 9JZ.
R. L. CUNDALL A. T. B. MOIR M. J. MARTIN.
1. Herzberg, L., Bold, A. M. Lancet, 1972, i, 1128. 2. Carney, M. W. P., Sheffield, B. F. ibid. p. 1287. 3. Cade, J. F. L. Med. J. Aust. 1964, i, 195. 4. Frizel, D., Coppen, A., Marks, V. Br, J, Psychiat. 1969, 115, 1375. 5. Bjørum, N. Acta psychiat. scand. 1972, 48, 59. 6. Cundall, R. L., Brooks, P. W., Murray, L. G. Psychol. Med. (in the press). 7. Birch, N. J. Br. J. Psychiat. 1970, 116, 461.
Hey, E., Hull, D. J. Obstet. Gynæc. Brit. Cwlth, 1971, 78, 1137. Bhagwanani, S. G., Fahmy, D., Turnbull, A. C. Lancet, 1972, i, 159. Whitfield, C. R., Chan, W. H., Sproule, W. B., Stewart, A. D. Br. med. J. 1972, ii, 85. 4. Lancet, 1971, ii, 477. 5. Prathap, K. ibid. 1972, i, 745. 6. Barva, R., Matthews, C. D. ibid. p. 956. 7. ibid. 1969, i, 32. 8. Scopes, J. W. Br. J. Hosp. Med. 1970, 3, 579. 9. Gregory, G. A., Kitterman, J. A., Roderick, M. D., Phibbs, H., Tooley, W. H., Hamilton, W. K. New Engl. J. Med. 1971, 284,
1. 2. 3.
1333. 10. Heitz, D. C.,
Jebson, P. J., Boutros, logy, 1971, 35, 61.
A.
R., Brody, M. J. Anesthesio-
282
vasoconstriction, provided the effects on the myocardium and peripheral circulation do not prevent a sufficient concentration of halothane being used. As with Dr. Prathap’s hypothesis,s these suggestions are theoretical but possibly worthy of trial. Department of Anesthetics, Royal Infirmary, Edinburgh EH3 9YW.
E. LL. LLOYD.
ASPIRIN AND SEMINAL PROSTAGLANDINS SIR,-Dr. Charles DeWitt Roberts (May 13, p. 1070) confirms statistically that aspirin reduces the concentration of prostaglandins E and F in seminal fluid.ll This mechanism is in accord with the effects of acetylsalicylic acid. This acid inhibits the release of histamine and S.R.S.12 (once thought to be prostaglandin) in the antigen/antibody reaction of anaphylaxis (now presumably IgE). I believe that aspirin inhibits the release of these substances by interfering with antibody groupings. 13 Further, I have shown 14 that aspirin also reduces the output of histamine in venom injury and that blood-probably its protein constituents -plays an essential part in the reaction. Spector and Willoughby 15 also implicated protein. It is possible that aspirin interferes with a membrane lipoprotein precursor releasing prostaglandin E and F. Department of Physiology, University of Melbourne, Australia.
E. R. TRETHEWIE.
HOSPITAL INFECTIONS CAUSED BY CONTAMINATED FLUIDS
SiR,-Dr. Dowsett’s interesting letter (June 17, p. 1338) did
whether the chlorhexidine solutions were analysed after autoclaving to check that the correct quantity of chlorhexidine survived the heat treatment. In 1964, at the Edinburgh Royal Infirmary, we made large batches of autoclaved, single use, 50 ml. bottles of aqueous 1/5000 chlorhexidine acetate and 10 oz. quantities of z20Savlon ’ solution. One of my quality-control pharmacists (Alan Hume) reported that analysis of certain bottles of the 1/5000 chlorhexidine-acetate solution showed 40% or more losses of chlorhexidine after autoclaving at 121°C for twenty minutes. It was found that the pH of distilled water (pH 6-0) rose to pH 9-5 after autoclaving in new glass lotion bottles at 121 °C for twenty minutes. It was found that the hot distilled water leached out alkali from the soft soda glass and this hot alkaline solution decomposed the ’Hibitane’. To neutralise this alkalinity we filled new bottles with 6% w/w acetic acid in freshly distilled water and, after autoclaving, rinsed them well with freshly distilled water and 1/5000 hibitane-acetate solution. 50 ml. of 1/5000 hibitane-acetate solution was filled into the bottles, which were sealed and re-autoclaved at 116°C for thirty minutes. The inner surface of these soda-glass bottles tends to break down after two or three autoclaving cycles and shed spicules of glass into the solutions. Attention is drawn to the need to analyse drug solutions after autoclaving. There are no alkali-free, boro-silicate glass lotion bottles manufactured in the United Kingdom. This lack of a range of heat-resistant hard-glass bottles suitable for autoclaving is a serious problem. South-Eastern Regional not state
Hospital Board, Scotland, Drumsheugh Gardens, Edinburgh EH3 7QQ.
HEPARIN EFFECTS ON IRREVERSIBLE PLATELET AGGREGATION SiR,—The letter by Dr. Eika (June 17, p. 1344) on heparin and platelets was of considerable interest to us and prompts us to report our results on the effects of heparin on irreversible platelet aggregation. Contrary to Dr. Eika’s findings, we have shown that heparin given intravenously or by in-vitro addition to citrated platelet-rich plasma (P.R.P.) resulted in the inhibition of irreversible platelet aggregation initiated by either collagen or adrenaline. Furthermore, our results suggest that the inhibitory effect of heparin in vivo is quite different from the direct inhibitory effect of high doses of heparin added to P.R.P. in vitro. In our in-vivo study of heparin effects on platelet aggregation we gave 2500 unit doses of mucous heparin (Weddel Pharmaceuticals Ltd.) intravenously to subjects undergoing routine right-heart catheterisation. Plateletcounts were not significantly different in P.R.P. obtained from blood samples taken before and 15 minutes after heparin was given. Irreversible platelet aggregation was compared in these paired P.R.P. samples which were tested under identical conditions, including preparation of P.R.P., platelet-counts, and relative times of testing. A range of concentrations of collagen or adrenaline were tested on pre-heparin P.R.P. and then repeated on postheparin samples of P.R.P. It was found that both the initial rate of aggregation and the extent (optical density change after 4 minutes) were reduced in post-heparin P.R.P. and that this difference was most evident at collagen or adrenaline concentrations which were just sufficient to cause maximum irreversible aggregation in the pre-
heparin The
P.R.P.
initial
rates
of
collagen-induced aggregation
15 minutes after the intravenous administration of 2500 units of heparin (mean values± standard deviation are shown for 11studies and the P value was calculated from Student’s t test) were:
immediately before and
11
11. 12. 13. 14. 15.
JOHN A. MYERS.
Collins, J. G., Flower, R. J. Lancet, 1971, ii, 852. Trethewie, E. R. Aust. J. exp. Biol. 1951, 29, 443. Trethewie, E. R. Med. J. Aust. 1952, i, 638. Trethewie, E. R., Morris, C. W. Aust. J. exp. Biol. 1959, 37, 567. Spector, W. G., Willoughby, D. A. J. Path. Bact. 1959, 78, 121.
Effect of
heparin added to platelet-rich plasma in vitro on collagen-induced aggregation. The percentage inhibition of aggregation was calculated from the comparison of the initial rates of aggregation of the heparintreated samples relative to that of a control sample containing no added heparin.
finding of paramyxovirus-like nucleocapsids in the tissueculture fluid (fig. 2). Fig. 3 shows a measles virus for comparison. The cytopathic agent was ether sensitive and resistant to 5-bromo-3-deoxy-uridine D.N.A. Although the finding of a paramyxovirus-like agent is of great interest it must be interpreted with considerable caution in view of well-recognised contamination dangers, especially when measles is in progress simultaneously. However, it is hoped that this brief report together with that of Dr. ter Meulen and his colleagues will encourage further efforts
at
virus isolation with newer methods. E. J. FIELD S. COWSHALL H. K. NARANG T. M. BELL.
M.R.C. Demyelinating Diseases Unit, Newcastle General Hospital,
Westgate Road, Newcastle upon Tyne NE4 6BE.
PLASMA AND ERYTHROCYTE MAGNESIUM LEVELS IN AFFECTIVE DISORDERS
SIR,-We should like to comment on the controversy magnesium levels in depression. Herzberg and Bold1 reported a significant difference in serum-magnesium levels between male and female depressed patients, but Carney and Sheffield2 could not confirm these results. Cade3 found raised serum levels during and after depression, whereas Frizel et awl. found the opposite-viz., serum-levels were low during depression and increased on recovery. Bjorumfound no difference between depressed and recovered patients, but the mean value in patients was higher than in controls. During a double-blind cross-over trial s of prophylactic lithium in affective disorders, samples of plasma and erythrocytes were analysed for magnesium, sodium, potassium, and lithium ions, using an atomic-absorption spectrophotometer (’ SP 90’, Pye Unicam). During the 6-month placebo period, samples (on average, three per patient) were taken every 1-2 months, from six male and nine female outpatients (the majority bipolar manic depressives). Preliminary analysis of our data reveals a significant difference in erythrocyte magnesium levels (expressed as mean±s.D. with number of observations in parentheses) between males, 4-77:0-45 meq. per litre (16), and females, 4-54:0-60 (25) (analysis of variance; P < 0-05). The mean plasma-magnesium in males, 1-890-21 (16), also seemed to be greater than in females, 1-82:0-15 (24), but this difference was not statistically significant. There were no significant differences in plasma or erythrocyte levels of sodium or potassium between males and females. Samples taken (on average, four per patient) from the same patients during 6 months on lithium carbonate showed no significant difference in lithium levels, in plasma or erythrocytes, between males and females. Lithium did not produce any significant alterations in plasma or erythrocyte levels of magnesium, sodium, or potassium. In view of the central role of magnesium in energyproducing enzyme reactionsthere is a need for further study of magnesium metabolism in affective disorders, with special reference to age and sex differences. over
One of
us
(R.
L.
C.)
was
supported by
a
Wellcome research
SALMONELLOSIS IN LIVESTOCK illusion about the ample evidence in the spread of salmonellosis are only too frequently reminded by the Public Health Laboratory Service of the human involvement in outbreaks of salmonella food-poisoning which regularly occur after wedding receptions and other such functions. I am sure both veterinary and medical personnel involved with the epidemiology of salmonellosis are fully aware of the animal origin of the causal organism and would totally disagree with Dr. Anderson’s playing-down of the human element in the spread of the disease. The investigation and control of any disease requires short-term immediate action which may be dispensed with once suitable long-term measures become available. As we know that salmonellas enter food establishments on some contaminated meat and poultry, is it too much to ask that the organisms be prevented from proliferating or spreading to wholesome food because of infected humans and poor hygienic control of food-handlers ? Would this be " wasted effort ", especially since salmonellosis in our animal population is not going to be controlled overnight.
SIR,-Ihave
no
incriminating humans (July 15, p. 138). We
University of Liverpool Department of Veterinary Medicine, Field Station, Leahurst, Neston,
JOHN
Wirral, Cheshire L64 7TE.
R. WALTON.
TREATMENT OF RESPIRATORY-DISTRESS SYNDROME
SIR,-Recent articles and correspondence in
your
journal suggest that the treatment of respiratory-distress syndrome of the newborn (R.D.S.) should be approached from a new angle. In the past, no treatment has been started until symptoms have developed, but new findings 1-3 suggest that the development of R.D.S. can be predicted at birth or even before. Therefore, if R.D.s. can be predicted, should start at birth to try to prevent the onset of symptoms, since it is generally accepted that R.D.s. is a self-limiting condition,4provided the child can be kept alive. The molecular oxygen in the inspired air may be toxic 5-6 and a raised " inspired " Po2 has many other dangers,7,8 but the arterial Po2 can be maintained with a lower inspired Po2 by continuous positive pressure applied to the airway,9 and use of this technique with an inspired Po2 of less than the 20% in normal air may be beneficial. Atelectasis and airway closure is one factor in the development of hypoxia 2,9 and, by substituting helium for nitrogen in the inspired gases, laminar flow may be preserved in some of the very small air passages, thus preventing atelectasis. Once hypoxia has developed, a vicious circle may be set up with the hypoxia causing pulmonary arteriolar constriction, resulting in further hypoxia.Halothane can relax the pulmonary circulation 10 and, by adding halothane to the inspired gases, a sufficient concentration may be achieved in the pulmonary vascular tree to counteract the hypoxic
treatment
fellowship in clinical pharmacolosv. M.R.C. Brain Metabolism Unit, University Department of Pharmacology, 1 George Square, Edinburgh EH8 9JZ.
R. L. CUNDALL A. T. B. MOIR M. J. MARTIN.
1. Herzberg, L., Bold, A. M. Lancet, 1972, i, 1128. 2. Carney, M. W. P., Sheffield, B. F. ibid. p. 1287. 3. Cade, J. F. L. Med. J. Aust. 1964, i, 195. 4. Frizel, D., Coppen, A., Marks, V. Br, J, Psychiat. 1969, 115, 1375. 5. Bjørum, N. Acta psychiat. scand. 1972, 48, 59. 6. Cundall, R. L., Brooks, P. W., Murray, L. G. Psychol. Med. (in the press). 7. Birch, N. J. Br. J. Psychiat. 1970, 116, 461.
Hey, E., Hull, D. J. Obstet. Gynæc. Brit. Cwlth, 1971, 78, 1137. Bhagwanani, S. G., Fahmy, D., Turnbull, A. C. Lancet, 1972, i, 159. Whitfield, C. R., Chan, W. H., Sproule, W. B., Stewart, A. D. Br. med. J. 1972, ii, 85. 4. Lancet, 1971, ii, 477. 5. Prathap, K. ibid. 1972, i, 745. 6. Barva, R., Matthews, C. D. ibid. p. 956. 7. ibid. 1969, i, 32. 8. Scopes, J. W. Br. J. Hosp. Med. 1970, 3, 579. 9. Gregory, G. A., Kitterman, J. A., Roderick, M. D., Phibbs, H., Tooley, W. H., Hamilton, W. K. New Engl. J. Med. 1971, 284,
1. 2. 3.
1333. 10. Heitz, D. C.,
Jebson, P. J., Boutros, logy, 1971, 35, 61.
A.
R., Brody, M. J. Anesthesio-
282
vasoconstriction, provided the effects on the myocardium and peripheral circulation do not prevent a sufficient concentration of halothane being used. As with Dr. Prathap’s hypothesis,s these suggestions are theoretical but possibly worthy of trial. Department of Anesthetics, Royal Infirmary, Edinburgh EH3 9YW.
E. LL. LLOYD.
ASPIRIN AND SEMINAL PROSTAGLANDINS SIR,-Dr. Charles DeWitt Roberts (May 13, p. 1070) confirms statistically that aspirin reduces the concentration of prostaglandins E and F in seminal fluid.ll This mechanism is in accord with the effects of acetylsalicylic acid. This acid inhibits the release of histamine and S.R.S.12 (once thought to be prostaglandin) in the antigen/antibody reaction of anaphylaxis (now presumably IgE). I believe that aspirin inhibits the release of these substances by interfering with antibody groupings. 13 Further, I have shown 14 that aspirin also reduces the output of histamine in venom injury and that blood-probably its protein constituents -plays an essential part in the reaction. Spector and Willoughby 15 also implicated protein. It is possible that aspirin interferes with a membrane lipoprotein precursor releasing prostaglandin E and F. Department of Physiology, University of Melbourne, Australia.
E. R. TRETHEWIE.
HOSPITAL INFECTIONS CAUSED BY CONTAMINATED FLUIDS
SiR,-Dr. Dowsett’s interesting letter (June 17, p. 1338) did
whether the chlorhexidine solutions were analysed after autoclaving to check that the correct quantity of chlorhexidine survived the heat treatment. In 1964, at the Edinburgh Royal Infirmary, we made large batches of autoclaved, single use, 50 ml. bottles of aqueous 1/5000 chlorhexidine acetate and 10 oz. quantities of z20Savlon ’ solution. One of my quality-control pharmacists (Alan Hume) reported that analysis of certain bottles of the 1/5000 chlorhexidine-acetate solution showed 40% or more losses of chlorhexidine after autoclaving at 121°C for twenty minutes. It was found that the pH of distilled water (pH 6-0) rose to pH 9-5 after autoclaving in new glass lotion bottles at 121 °C for twenty minutes. It was found that the hot distilled water leached out alkali from the soft soda glass and this hot alkaline solution decomposed the ’Hibitane’. To neutralise this alkalinity we filled new bottles with 6% w/w acetic acid in freshly distilled water and, after autoclaving, rinsed them well with freshly distilled water and 1/5000 hibitane-acetate solution. 50 ml. of 1/5000 hibitane-acetate solution was filled into the bottles, which were sealed and re-autoclaved at 116°C for thirty minutes. The inner surface of these soda-glass bottles tends to break down after two or three autoclaving cycles and shed spicules of glass into the solutions. Attention is drawn to the need to analyse drug solutions after autoclaving. There are no alkali-free, boro-silicate glass lotion bottles manufactured in the United Kingdom. This lack of a range of heat-resistant hard-glass bottles suitable for autoclaving is a serious problem. South-Eastern Regional not state
Hospital Board, Scotland, Drumsheugh Gardens, Edinburgh EH3 7QQ.
HEPARIN EFFECTS ON IRREVERSIBLE PLATELET AGGREGATION SiR,—The letter by Dr. Eika (June 17, p. 1344) on heparin and platelets was of considerable interest to us and prompts us to report our results on the effects of heparin on irreversible platelet aggregation. Contrary to Dr. Eika’s findings, we have shown that heparin given intravenously or by in-vitro addition to citrated platelet-rich plasma (P.R.P.) resulted in the inhibition of irreversible platelet aggregation initiated by either collagen or adrenaline. Furthermore, our results suggest that the inhibitory effect of heparin in vivo is quite different from the direct inhibitory effect of high doses of heparin added to P.R.P. in vitro. In our in-vivo study of heparin effects on platelet aggregation we gave 2500 unit doses of mucous heparin (Weddel Pharmaceuticals Ltd.) intravenously to subjects undergoing routine right-heart catheterisation. Plateletcounts were not significantly different in P.R.P. obtained from blood samples taken before and 15 minutes after heparin was given. Irreversible platelet aggregation was compared in these paired P.R.P. samples which were tested under identical conditions, including preparation of P.R.P., platelet-counts, and relative times of testing. A range of concentrations of collagen or adrenaline were tested on pre-heparin P.R.P. and then repeated on postheparin samples of P.R.P. It was found that both the initial rate of aggregation and the extent (optical density change after 4 minutes) were reduced in post-heparin P.R.P. and that this difference was most evident at collagen or adrenaline concentrations which were just sufficient to cause maximum irreversible aggregation in the pre-
heparin The
P.R.P.
initial
rates
of
collagen-induced aggregation
15 minutes after the intravenous administration of 2500 units of heparin (mean values± standard deviation are shown for 11studies and the P value was calculated from Student’s t test) were:
immediately before and
11
11. 12. 13. 14. 15.
JOHN A. MYERS.
Collins, J. G., Flower, R. J. Lancet, 1971, ii, 852. Trethewie, E. R. Aust. J. exp. Biol. 1951, 29, 443. Trethewie, E. R. Med. J. Aust. 1952, i, 638. Trethewie, E. R., Morris, C. W. Aust. J. exp. Biol. 1959, 37, 567. Spector, W. G., Willoughby, D. A. J. Path. Bact. 1959, 78, 121.
Effect of
heparin added to platelet-rich plasma in vitro on collagen-induced aggregation. The percentage inhibition of aggregation was calculated from the comparison of the initial rates of aggregation of the heparintreated samples relative to that of a control sample containing no added heparin.