- Email: [email protected]
HEPARIN EFFECTS ON IRREVERSIBLE PLATELET AGGREGATION
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.
283
of aggregation was reduced in post-heparin P.R.P. By contrast, the rate and extent of reversible aggregation induced by adenosine diphosphate (A.D.P.) was usually slightly stimulated in post-heparin P.R.P. When heparin was added to citrated P.R.P. in vitro at concentrations similar to those pertaining to our in-vivo study (0’5-1 unit per ml.), it had no effect on collageninduced aggregation. However, when P.R.P. was incubated with increasing concentrations of heparin, then both the rate and extent of collagen-induced aggregation were reduced, indicating that at these concentrations heparin has a direct inhibitory effect on collagen-induced aggregation (see accompanying figure). This confirms the earlier report from O’Brien et al.l that heparin in vitro inhibited
phase
LEUCOCYTE FUNCTION IN DOWN’S SYNDROME
SIR,-Dr. Gregory and her colleagues (June 24, p. 1359) reported a high frequency of partial leucocyte dysfunction against staphylococci in children with Down’s syndrome and in children with acute leukxmia. We have described1 abnormal leucocyte function in patients with acute and chronic leuksmia. We have studied the in-vitro phagocytic ability of peripheral-blood neutrophils to ingest live Candida albicans, and neutrophil adhesiveness in patients with Down’s syndrome. They were significantly decreased in the patients with Down’s syndrome as compared with those of control subjects:
irreversible
platelet aggregation. these inhibitory concentrations of heparin in vitro were far higher than those to be expected after the intravenous administration of only 2500 units of heparin. However,
Thus, the decreased irreversible aggregation which we have found in vivo was probably due to an indirect action of heparin in vivo. Intravenous heparin (2500 units) increases the rate of lysolecithin formation in incubated human plasma,2and lysolecithin added to P.R.P. in vitro inhibits irreversible aggregation initiated by adrenaline, collagen, or A.D.P.3 It is tempting to speculate that the reduction in collagen and adrenaline induced irreversible platelet aggregation following intravenous heparin may result from this concomitant increase in lysolecithin formation.
Department of Cardiology, St. Mary’s Hospital, London W.2.
E. M. M. BESTERMAN M. P. T. GILLETT.
ANÆMIA WITH ARTIFICIAL HEART-VALVES SIR,-Your editorial on this topic (June 17, p. 1321) rightly drew attention to the significance of urinary iron loss. There is no doubt that in both severe and compensated cardiac hsemolytic ansmia there is loss of iron in the urine. We have also observed hypochromia and low serumiron levels in many of our patients with prosthetic heartvalves. We agree that it is essential to examine the urine of all patients with prosthetic valves at regular intervals for the presence of haemosiderin. As soon as hsemosiderin is detected, oral iron should be prescribed. Iron therapy should be continued indefinitely, since the haanosiderinuria is usually persistent and may lead eventually to iron deficiency. The absence of anxmia does not exclude
haemolysis, since patients with significant hxnlolysis
can
maintain normal hamiatocrits by increasing erythrocyte production, sometimes as much as eightfold.44 This accelerated rate of production can be maintained only as long as an adequate supply of iron is available. At the moment we are comparing the frequency and severity of haemolysis of the Starr-Edwards prosthesis in the mitral position with that of the mounted aortic homograft in the same position. Our preliminary results support the findings of Slater and Fell,5 in that 60% of our patients with Starr valves in the mitral position have hsemosiderinuria, but none of the patients with homografts in the mitral position have haemosiderin in the urine. Leeds Regional Thoracic
Surgical Centre, Killingbeck Hospital, York Road, Leeds LS14 6UQ. 1. 2. 3. 4. 5.
R. J. DONNELLY A. N. RAHMAN
S. M. MANOHITHARAJAH D. A. WATSON.
O’Brien, J. R., Shoobridge, S. M., Finch, W. J. J. clin. Path. 1969, 22, 28. Gillett, M. P. T. Clin. Sci. 1972, 43, 1P. Besterman, E. M. M., Gillett, M. P. T. Atherosclerosis, 1971, 14, 323. Hillman, R. S., Henderson, P. A. J. clin. Invest. 1969, 48, 454. Slater, S. D., Fell, G. S. Clin. Sci. 1972, 42, 545.
Peroxidase and periodic-acid/Schiff activity in leucocytes was normal. Humoral immunity was assessed by quantitative measurement of serum-immunoglobulins. IgM levels were normal, IgG levels were increased, and IgA levels were increased only in adults (above 18 years of age) with Down’s syndrome :
Antinuclear antibodies were not present in any Down’s patient, and serum-haptoglobin levels were normal. Two patients had high-titre rheumatoid factor. Our results indicate that leucocyte function as measured by phagocytic ability and adhesiveness index is decreased in patients with Down’s syndrome. Previous work 2,3 indicated a variety of biochemical abnormalities in the leucocytes of such patients. Myeloperoxidase deficiency has been reported 4 to be associated with defective candidacidal activity in leucocytes and apparent enhanced susceptibility In the present study, to systemic candida infection. peroxidase in leucocytes was found to be normal. Furthermore, the recent finding of increased leucocyte glycogen,5 as measured by the periodic-acid/Schiff reaction, could not be confirmed. Whether these biochemical aberrations are related to the decreased phagocytic ability of leucocytes and the decreased adhesiveness of leucocytes is not known. It is also not clear whether abnormalities in leucocyte 1. Rosner, F., Valmont, I., Kozinn, P. J., Caroline, L. Cancer, 1970, 25, 835. 2. Rosner, F., Ong, B. H., Paine, R. S., Mahanand, D. New Engl. J. Med. 1965, 273, 1356. 3. Hsia, D. Y., Justice, P., Smith, G. F., Dowben, R. M. Am. J. Dis. Child. 1971, 121, 153. 4. Salmon, S. E., Cline, M. J., Schultz, J., Lehrer, R. I. New Engl. J. Med. 1970, 282, 250. 5. Abul-Fadl, Y., Scott, R. B. Nature, 1969, 223, 310.
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.
283
of aggregation was reduced in post-heparin P.R.P. By contrast, the rate and extent of reversible aggregation induced by adenosine diphosphate (A.D.P.) was usually slightly stimulated in post-heparin P.R.P. When heparin was added to citrated P.R.P. in vitro at concentrations similar to those pertaining to our in-vivo study (0’5-1 unit per ml.), it had no effect on collageninduced aggregation. However, when P.R.P. was incubated with increasing concentrations of heparin, then both the rate and extent of collagen-induced aggregation were reduced, indicating that at these concentrations heparin has a direct inhibitory effect on collagen-induced aggregation (see accompanying figure). This confirms the earlier report from O’Brien et al.l that heparin in vitro inhibited
phase
LEUCOCYTE FUNCTION IN DOWN’S SYNDROME
SIR,-Dr. Gregory and her colleagues (June 24, p. 1359) reported a high frequency of partial leucocyte dysfunction against staphylococci in children with Down’s syndrome and in children with acute leukxmia. We have described1 abnormal leucocyte function in patients with acute and chronic leuksmia. We have studied the in-vitro phagocytic ability of peripheral-blood neutrophils to ingest live Candida albicans, and neutrophil adhesiveness in patients with Down’s syndrome. They were significantly decreased in the patients with Down’s syndrome as compared with those of control subjects:
irreversible
platelet aggregation. these inhibitory concentrations of heparin in vitro were far higher than those to be expected after the intravenous administration of only 2500 units of heparin. However,
Thus, the decreased irreversible aggregation which we have found in vivo was probably due to an indirect action of heparin in vivo. Intravenous heparin (2500 units) increases the rate of lysolecithin formation in incubated human plasma,2and lysolecithin added to P.R.P. in vitro inhibits irreversible aggregation initiated by adrenaline, collagen, or A.D.P.3 It is tempting to speculate that the reduction in collagen and adrenaline induced irreversible platelet aggregation following intravenous heparin may result from this concomitant increase in lysolecithin formation.
Department of Cardiology, St. Mary’s Hospital, London W.2.
E. M. M. BESTERMAN M. P. T. GILLETT.
ANÆMIA WITH ARTIFICIAL HEART-VALVES SIR,-Your editorial on this topic (June 17, p. 1321) rightly drew attention to the significance of urinary iron loss. There is no doubt that in both severe and compensated cardiac hsemolytic ansmia there is loss of iron in the urine. We have also observed hypochromia and low serumiron levels in many of our patients with prosthetic heartvalves. We agree that it is essential to examine the urine of all patients with prosthetic valves at regular intervals for the presence of haemosiderin. As soon as hsemosiderin is detected, oral iron should be prescribed. Iron therapy should be continued indefinitely, since the haanosiderinuria is usually persistent and may lead eventually to iron deficiency. The absence of anxmia does not exclude
haemolysis, since patients with significant hxnlolysis
can
maintain normal hamiatocrits by increasing erythrocyte production, sometimes as much as eightfold.44 This accelerated rate of production can be maintained only as long as an adequate supply of iron is available. At the moment we are comparing the frequency and severity of haemolysis of the Starr-Edwards prosthesis in the mitral position with that of the mounted aortic homograft in the same position. Our preliminary results support the findings of Slater and Fell,5 in that 60% of our patients with Starr valves in the mitral position have hsemosiderinuria, but none of the patients with homografts in the mitral position have haemosiderin in the urine. Leeds Regional Thoracic
Surgical Centre, Killingbeck Hospital, York Road, Leeds LS14 6UQ. 1. 2. 3. 4. 5.
R. J. DONNELLY A. N. RAHMAN
S. M. MANOHITHARAJAH D. A. WATSON.
O’Brien, J. R., Shoobridge, S. M., Finch, W. J. J. clin. Path. 1969, 22, 28. Gillett, M. P. T. Clin. Sci. 1972, 43, 1P. Besterman, E. M. M., Gillett, M. P. T. Atherosclerosis, 1971, 14, 323. Hillman, R. S., Henderson, P. A. J. clin. Invest. 1969, 48, 454. Slater, S. D., Fell, G. S. Clin. Sci. 1972, 42, 545.
Peroxidase and periodic-acid/Schiff activity in leucocytes was normal. Humoral immunity was assessed by quantitative measurement of serum-immunoglobulins. IgM levels were normal, IgG levels were increased, and IgA levels were increased only in adults (above 18 years of age) with Down’s syndrome :
Antinuclear antibodies were not present in any Down’s patient, and serum-haptoglobin levels were normal. Two patients had high-titre rheumatoid factor. Our results indicate that leucocyte function as measured by phagocytic ability and adhesiveness index is decreased in patients with Down’s syndrome. Previous work 2,3 indicated a variety of biochemical abnormalities in the leucocytes of such patients. Myeloperoxidase deficiency has been reported 4 to be associated with defective candidacidal activity in leucocytes and apparent enhanced susceptibility In the present study, to systemic candida infection. peroxidase in leucocytes was found to be normal. Furthermore, the recent finding of increased leucocyte glycogen,5 as measured by the periodic-acid/Schiff reaction, could not be confirmed. Whether these biochemical aberrations are related to the decreased phagocytic ability of leucocytes and the decreased adhesiveness of leucocytes is not known. It is also not clear whether abnormalities in leucocyte 1. Rosner, F., Valmont, I., Kozinn, P. J., Caroline, L. Cancer, 1970, 25, 835. 2. Rosner, F., Ong, B. H., Paine, R. S., Mahanand, D. New Engl. J. Med. 1965, 273, 1356. 3. Hsia, D. Y., Justice, P., Smith, G. F., Dowben, R. M. Am. J. Dis. Child. 1971, 121, 153. 4. Salmon, S. E., Cline, M. J., Schultz, J., Lehrer, R. I. New Engl. J. Med. 1970, 282, 250. 5. Abul-Fadl, Y., Scott, R. B. Nature, 1969, 223, 310.