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INCREASE IN PLATELET AGGREGATION WITH TIME AFTER VENEPUNCTURE
893 INCREASE IN PLATELET AGGREGATION WITH TIME AFTER VENEPUNCTURE SIR,-Dr. Fyfe and Eleanor Hamilton (Sept. 9, p. 542) found significant progressive increase in platelet adhesiveness with time after venepunture ". We found a similar increase in platelet aggregation when this was studied by our method, soon to be published, which seems to provide an explanation. With a model A Coulter counter we determined the particle content of citrated or heparinised platelet-rich plasma (P.R.P.) before and after addition of aggregating agents such as adenosine diphosphate (A.D.P.) and noradrenaline. We found that P.R.P., after standing at room-temperature or after much handling, spontaneously showed a drop in total particle count and an increase of larger-sized particles (see accompanying table). The "
DUPLICATE PARTICLE-COUNTS
20° C,
Percentages of diploid metaphases in three groups of mice: normal, and 24 hours after injection with normal human plasma, and with human leukaemic plasma. Human blood was obtained with sterile precautions, using 3-8% sodium-citrate solution as anticoagulant. The plasma was separated by centrifugation at 800 r.p.m. for 8 minutes, and the upper twothirds of the supernatant was pipetted and centrifugated again at the
speed for 10 minutes; each mouse was injected intraperitoneally with 0-5 ml. from the upper two-thirds of this portion. No cells were seen in samples of the injected material. Chromosomes were studied in three groups of animals: (a) 27 normal mice; (b) 11mice injected with normal human plasma; (c) 17 mice injected with plasma from three adult patients with acute leukaemia (two with untreated myeloblastic leukaemia, and one with undifferentiated leukaemia, treated with corticosteroids, who had received 700 mg. of mercaptopurine two months before). In groups (b) and (c) the colchicine was injected 24 hours after the humansame
(per l.)
IN CITRATED
P.R.P., KEPT
AT
WITH A COULTER COUNTER
drop in particle count in P.R.P. at room-temperature nearly completely reversed when the plasma-temperature was brought up to 37°C. We also found increased aggregation by A.D.P. with time after venepunture (see accompanying figure). Blood anticoagulated with edeticacid (E.D.T.A.), which is of course not suited for A.D.P.-aggregation, did not show this spontaneous drop of particle count. These observations show that " spontaneous " aggregation occurs during standing of blood or P.R.P. Aggregation by A.D.p.-addition increased simultaneously with this " spontaneous " aggregation. Possibly this increase is caused by release of A.D.P. from the platelets during standing.
Glynn
et
plasma injection. In group (a) an average of 96% of the 720 metaphases were normal. The results of group (b), which were not significantly different from those of group (a), showed that an average of 96% of the 469 metaphases counted were normal. The results in group (c), however, were completely different from those in both the previous groups. Of the 821 metaphases counted the percentage of euploid metaphases fell strikingly to 75%, the percentage of hypodiploid and hyperdiploid metaphases was increased. The accompanying figure shows that most of the counts in this group were under 80% while in groups (a) and (b) they were over 90%. No constant structural chromosomal were identified. These abnormal findings in the hxmatopoietic cells of normal mice injected with human leukxmic plasma suggest a hypothetic plasmatic factor in this illness, which affects the mechanism of cellular division in its early stages. The following questions arise: (1) Is the action of this factor transitory or persistent ? Initial data gathered at our laboratory show that the affected cells gradually become normal; (2) Is it viral or humoral in nature ? ; (3) Is it specific for leukaemia ?; (4) What is its pathogenic significance in patients with leukxmia? New
changes
techniques
are
investigate
some
now
being developed points.
at our
laboratory
to
of these
We thank Dr. J. Ferrer for advice, and Mrs. H. T. Scarponi and Mrs. A. M. Otazu for their assistance.
Pathology Department, School of Biochemistry, Faculty of Medicine, Rosario, Argentina.
ISAAC SCHLAEN NORBERTO V. GILLIAVOD HELIO VAZQUEZ.
Arrows show drop in number of non-aggregated 5 minutes after addition of A.D.P.
platelets within
Coulter counter at different thresholds, particles was subtracted from the total particle count. P.R.P. was kept at 20°C. Platelet-count in thousands per 1. P.R.P.
Counts were made with and the number of large End concentrations of 0-25 .g. per ml.
a
A.D.P.
0-50 jjg. per ml.
and
894 aLl and Mustard et al. have demonstrated such a release from platelets incubated with latex particles, antigen-antibody complexes, uric-acid crystals, and other foreign substances. It seems logical to conclude that " spontaneous " aggregation may play a principal role in the reported increase in adhesiveness during standing of blood when using Payling Wright’s technique
Division of
Hæmatology, Department of Medicine, St. Radboud Hospital, University of Nijmegen, The Netherlands.
A. HOLDRINET M. EWALS C. HAANEN.
Furthermore, the total white-blood-cell counts in in group II decreased rapidly during the first ten days of the experiment from a mean of 7000 per
the rabbits to
fourteen
c.mm. to a
None of the rabbits in groups I and III displayed depression in their circulating white-cell counts. No further deaths were recorded between days twenty and sixty, when the experiment was terminated. These observations suggest that P.H.A. may be a useful drug in counteracting the debilitating, leucopenic, and lethal effects of overexuberant immunosuppressant therapy. This study was supported by a grant from the Medical Research
mean
of 2000 per
c.mm.
any
Council, Canada. MEGALOBLASTIC ANÆMIA AND VISION SIR,-We are grateful to Dr. Freeman (Aug. 19, p. 422) and Mr. Gorell (Aug. 26, p. 469) for their interesting comments on our paper. We agree with Mr. Gorell that in any group of elderly patients some are likely to show alterations in colour discrimination owing to yellow pigmentation of the lens and for other reasons. However, a defect of colour discrimination would be unlikely to disappear on treatment of a coexisting anaemia, unless the two abnormalities were in some way related. Our further work shows that a severe reversible defect of colour discrimination is common in patients with megaloblastic anasmia, and that it can occur in younger patients and in patients with vitamin-B12 deficiency who are not anaemic. While the defect tends to lie on the yellow-blue axis, some loss in the red-green axis, detectable by Ishihara pseudo-isochromatic plates, is also usual. This general reduction in colour discrimination differs from the striking red/green loss which occurs in the rare optic neuropathy of patients with pernicious anaemia, an example of which was described in our article rTulv 29.
n
Harry Webster Thorp Laboratories, Division of Immunochemistry and Allergy, McGill University Clinic, Royal Victoria Hospital, Montreal, Quebec, Canada.
MAXWELL RICHTER MICHAEL MANDL.
HÆMOLYTIC-ANÆMIA RELAPSES AFTER IMMUNISATION AND PERTUSSIS SIR,-Autoimmune h2emolytic anaemia (A.H.A.) has been reported after virus disease or bacterial infections such as typhoid fever.1 Possible implication of viruses such as influenza virus A, Coxsackie virus A,l and viruses of measles,2 and varicella3 has also been reported. There are also reports of apparent initiation of exacerbation of this ansemia after vaccination against smallpox 4 or poliomyelitis.5 We describe here a patient with severe relapses of A.H.A. after diphtheria-tetanustyphoid immunisation and pertussis infection. The patient was a six-year-old Caucasian school-boy in whom A.H.A. had been diagnosed in March, 1965, and who had been treated with prednisolone (see accompanying table). He
229).
Addenbrooke’s Hospital, Hills Road, Cambridge.
PETER ADAMS T. M. CHALMERS W. S. FOULDS J. L. WITHEY.
IMMUNOSUPPRESSION AND PHYTOHÆMAGGLUTININ SIR,-It has been demonstrated that adult rabbits can be made immunologically tolerant to protein antigens provided they are injected with an immunosuppressant-i.e., 6-mercaptopurine (6-M.p.)-for a period of several weeks following the administration of the antigen.3 A derivative of 6-M.P., immuran, is also used in man to facilitate acceptance of homografts, such as kidney. However, many transplanted individuals subsequently die, not as a result of rejection of the transplanted kidney, but as a result of septicxmia and leucopenia, probably facilitated by overdosage of immunosuppressant. We have initiated an investigation into the use of phytohaemagglutinin (P.H.A.) as an agent which may reverse or inhibit the debilitating and lethal effects of immuran. 11 adult rabbits (group I) were injected daily subcutaneously with 6-M.P. (Burroughs Wellcome) (10 mg. per kg. body-weight per day) and intravenously with 1/4 bottleP.H.A.-M’ (Difco) per day for fourteen days. 18 other rabbits (group n) were injected daily with 6-M.P. (10 mg. per kg. body-weight daily) for fourteen days, and 10 rabbits (group III) were injected with P.H.A. only’ (1/4 bottle P.H.A.-M per day) for fourteen days. By day twenty, 10 of the rabbits (60%) in group II had died, whereas only 1 (10%) in group I and none in group III had died. In fact, all the surviving rabbits in group I looked healthy with no sign of stress. All the rabbits in group 11 had lost 25-30% of their weights by day twenty, whereas all the rabbits in groups I and III either lost no weight or gained weight. 1. Glynn, M. F., Packham, M. A., Hirsh, J., Mustard, J. F. J. clin. Invest. 1966, 45, 1013. 2. Mustard, J. F., Glynn, M. F., Nishizawa, E. E., Packham, M. A., Fedn Proc. Fedn Am. Socs exp. Biol. 1967, 26, 106. 3. Schwartz, R. S., Dameshek, W. J. Immun. 1963, 90, 703.
Betke, K., Richarz, H., Schubothe, H., Vivell, O. Klin. Wschr. 1953, 31, 373. 2. Dacie, J. V. The Hæmolytic Anæmias. Congenital and Acquired. London, 1954. 3. Borbella, L., Barquet Chediak, A. Revla Cub. Pediat. 1953, 25, 557. 4. Dacie, J. V. The Hæmolytic Anæmias. Part II. The Autoimmune Hæmolytic Anæmias; chap. 9. London, 1962. 5. Laroche, C., Milliez, P., Dreyfus, B., Dausset, J., Leprat, J. Bull. Mém. Soc. méd. Hôp. Paris, 1951, 67, 779. 1.
BLOOD AND BONE-MARROW FINDINGS IN PATIENT WITH A.H.A. DURING 3 ADMISSIONS TO HOSPITAL BEFORE AND AFTER TREATMENT WITH PREDNISOLONE
.Treatment: prednisolone (’ Medrol22 mg. per kg. per day for three tapering off. Erythrocyte glucose-6-phosphate-dehydrogenase activity normal, test for haemoglobin F negative, serum-iron 77-0 tig. per 100 ml., serum-glutamic-
weeks and then
pyruvic-transaminase 20 i.u., serum-glutamic-oxaloacetic-transaminase 26 i.u., serum-lactic-acid-dehydrogenase 1500 units, and bone-marrow compatible with A.H.A., on first admission to hospital (before treatment). *
After
D.T.T.
immunisation.
t During pertussis infection.
DUPLICATE PARTICLE-COUNTS
20° C,
Percentages of diploid metaphases in three groups of mice: normal, and 24 hours after injection with normal human plasma, and with human leukaemic plasma. Human blood was obtained with sterile precautions, using 3-8% sodium-citrate solution as anticoagulant. The plasma was separated by centrifugation at 800 r.p.m. for 8 minutes, and the upper twothirds of the supernatant was pipetted and centrifugated again at the
speed for 10 minutes; each mouse was injected intraperitoneally with 0-5 ml. from the upper two-thirds of this portion. No cells were seen in samples of the injected material. Chromosomes were studied in three groups of animals: (a) 27 normal mice; (b) 11mice injected with normal human plasma; (c) 17 mice injected with plasma from three adult patients with acute leukaemia (two with untreated myeloblastic leukaemia, and one with undifferentiated leukaemia, treated with corticosteroids, who had received 700 mg. of mercaptopurine two months before). In groups (b) and (c) the colchicine was injected 24 hours after the humansame
(per l.)
IN CITRATED
P.R.P., KEPT
AT
WITH A COULTER COUNTER
drop in particle count in P.R.P. at room-temperature nearly completely reversed when the plasma-temperature was brought up to 37°C. We also found increased aggregation by A.D.P. with time after venepunture (see accompanying figure). Blood anticoagulated with edeticacid (E.D.T.A.), which is of course not suited for A.D.P.-aggregation, did not show this spontaneous drop of particle count. These observations show that " spontaneous " aggregation occurs during standing of blood or P.R.P. Aggregation by A.D.p.-addition increased simultaneously with this " spontaneous " aggregation. Possibly this increase is caused by release of A.D.P. from the platelets during standing.
Glynn
et
plasma injection. In group (a) an average of 96% of the 720 metaphases were normal. The results of group (b), which were not significantly different from those of group (a), showed that an average of 96% of the 469 metaphases counted were normal. The results in group (c), however, were completely different from those in both the previous groups. Of the 821 metaphases counted the percentage of euploid metaphases fell strikingly to 75%, the percentage of hypodiploid and hyperdiploid metaphases was increased. The accompanying figure shows that most of the counts in this group were under 80% while in groups (a) and (b) they were over 90%. No constant structural chromosomal were identified. These abnormal findings in the hxmatopoietic cells of normal mice injected with human leukxmic plasma suggest a hypothetic plasmatic factor in this illness, which affects the mechanism of cellular division in its early stages. The following questions arise: (1) Is the action of this factor transitory or persistent ? Initial data gathered at our laboratory show that the affected cells gradually become normal; (2) Is it viral or humoral in nature ? ; (3) Is it specific for leukaemia ?; (4) What is its pathogenic significance in patients with leukxmia? New
changes
techniques
are
investigate
some
now
being developed points.
at our
laboratory
to
of these
We thank Dr. J. Ferrer for advice, and Mrs. H. T. Scarponi and Mrs. A. M. Otazu for their assistance.
Pathology Department, School of Biochemistry, Faculty of Medicine, Rosario, Argentina.
ISAAC SCHLAEN NORBERTO V. GILLIAVOD HELIO VAZQUEZ.
Arrows show drop in number of non-aggregated 5 minutes after addition of A.D.P.
platelets within
Coulter counter at different thresholds, particles was subtracted from the total particle count. P.R.P. was kept at 20°C. Platelet-count in thousands per 1. P.R.P.
Counts were made with and the number of large End concentrations of 0-25 .g. per ml.
a
A.D.P.
0-50 jjg. per ml.
and
894 aLl and Mustard et al. have demonstrated such a release from platelets incubated with latex particles, antigen-antibody complexes, uric-acid crystals, and other foreign substances. It seems logical to conclude that " spontaneous " aggregation may play a principal role in the reported increase in adhesiveness during standing of blood when using Payling Wright’s technique
Division of
Hæmatology, Department of Medicine, St. Radboud Hospital, University of Nijmegen, The Netherlands.
A. HOLDRINET M. EWALS C. HAANEN.
Furthermore, the total white-blood-cell counts in in group II decreased rapidly during the first ten days of the experiment from a mean of 7000 per
the rabbits to
fourteen
c.mm. to a
None of the rabbits in groups I and III displayed depression in their circulating white-cell counts. No further deaths were recorded between days twenty and sixty, when the experiment was terminated. These observations suggest that P.H.A. may be a useful drug in counteracting the debilitating, leucopenic, and lethal effects of overexuberant immunosuppressant therapy. This study was supported by a grant from the Medical Research
mean
of 2000 per
c.mm.
any
Council, Canada. MEGALOBLASTIC ANÆMIA AND VISION SIR,-We are grateful to Dr. Freeman (Aug. 19, p. 422) and Mr. Gorell (Aug. 26, p. 469) for their interesting comments on our paper. We agree with Mr. Gorell that in any group of elderly patients some are likely to show alterations in colour discrimination owing to yellow pigmentation of the lens and for other reasons. However, a defect of colour discrimination would be unlikely to disappear on treatment of a coexisting anaemia, unless the two abnormalities were in some way related. Our further work shows that a severe reversible defect of colour discrimination is common in patients with megaloblastic anasmia, and that it can occur in younger patients and in patients with vitamin-B12 deficiency who are not anaemic. While the defect tends to lie on the yellow-blue axis, some loss in the red-green axis, detectable by Ishihara pseudo-isochromatic plates, is also usual. This general reduction in colour discrimination differs from the striking red/green loss which occurs in the rare optic neuropathy of patients with pernicious anaemia, an example of which was described in our article rTulv 29.
n
Harry Webster Thorp Laboratories, Division of Immunochemistry and Allergy, McGill University Clinic, Royal Victoria Hospital, Montreal, Quebec, Canada.
MAXWELL RICHTER MICHAEL MANDL.
HÆMOLYTIC-ANÆMIA RELAPSES AFTER IMMUNISATION AND PERTUSSIS SIR,-Autoimmune h2emolytic anaemia (A.H.A.) has been reported after virus disease or bacterial infections such as typhoid fever.1 Possible implication of viruses such as influenza virus A, Coxsackie virus A,l and viruses of measles,2 and varicella3 has also been reported. There are also reports of apparent initiation of exacerbation of this ansemia after vaccination against smallpox 4 or poliomyelitis.5 We describe here a patient with severe relapses of A.H.A. after diphtheria-tetanustyphoid immunisation and pertussis infection. The patient was a six-year-old Caucasian school-boy in whom A.H.A. had been diagnosed in March, 1965, and who had been treated with prednisolone (see accompanying table). He
229).
Addenbrooke’s Hospital, Hills Road, Cambridge.
PETER ADAMS T. M. CHALMERS W. S. FOULDS J. L. WITHEY.
IMMUNOSUPPRESSION AND PHYTOHÆMAGGLUTININ SIR,-It has been demonstrated that adult rabbits can be made immunologically tolerant to protein antigens provided they are injected with an immunosuppressant-i.e., 6-mercaptopurine (6-M.p.)-for a period of several weeks following the administration of the antigen.3 A derivative of 6-M.P., immuran, is also used in man to facilitate acceptance of homografts, such as kidney. However, many transplanted individuals subsequently die, not as a result of rejection of the transplanted kidney, but as a result of septicxmia and leucopenia, probably facilitated by overdosage of immunosuppressant. We have initiated an investigation into the use of phytohaemagglutinin (P.H.A.) as an agent which may reverse or inhibit the debilitating and lethal effects of immuran. 11 adult rabbits (group I) were injected daily subcutaneously with 6-M.P. (Burroughs Wellcome) (10 mg. per kg. body-weight per day) and intravenously with 1/4 bottleP.H.A.-M’ (Difco) per day for fourteen days. 18 other rabbits (group n) were injected daily with 6-M.P. (10 mg. per kg. body-weight daily) for fourteen days, and 10 rabbits (group III) were injected with P.H.A. only’ (1/4 bottle P.H.A.-M per day) for fourteen days. By day twenty, 10 of the rabbits (60%) in group II had died, whereas only 1 (10%) in group I and none in group III had died. In fact, all the surviving rabbits in group I looked healthy with no sign of stress. All the rabbits in group 11 had lost 25-30% of their weights by day twenty, whereas all the rabbits in groups I and III either lost no weight or gained weight. 1. Glynn, M. F., Packham, M. A., Hirsh, J., Mustard, J. F. J. clin. Invest. 1966, 45, 1013. 2. Mustard, J. F., Glynn, M. F., Nishizawa, E. E., Packham, M. A., Fedn Proc. Fedn Am. Socs exp. Biol. 1967, 26, 106. 3. Schwartz, R. S., Dameshek, W. J. Immun. 1963, 90, 703.
Betke, K., Richarz, H., Schubothe, H., Vivell, O. Klin. Wschr. 1953, 31, 373. 2. Dacie, J. V. The Hæmolytic Anæmias. Congenital and Acquired. London, 1954. 3. Borbella, L., Barquet Chediak, A. Revla Cub. Pediat. 1953, 25, 557. 4. Dacie, J. V. The Hæmolytic Anæmias. Part II. The Autoimmune Hæmolytic Anæmias; chap. 9. London, 1962. 5. Laroche, C., Milliez, P., Dreyfus, B., Dausset, J., Leprat, J. Bull. Mém. Soc. méd. Hôp. Paris, 1951, 67, 779. 1.
BLOOD AND BONE-MARROW FINDINGS IN PATIENT WITH A.H.A. DURING 3 ADMISSIONS TO HOSPITAL BEFORE AND AFTER TREATMENT WITH PREDNISOLONE
.Treatment: prednisolone (’ Medrol22 mg. per kg. per day for three tapering off. Erythrocyte glucose-6-phosphate-dehydrogenase activity normal, test for haemoglobin F negative, serum-iron 77-0 tig. per 100 ml., serum-glutamic-
weeks and then
pyruvic-transaminase 20 i.u., serum-glutamic-oxaloacetic-transaminase 26 i.u., serum-lactic-acid-dehydrogenase 1500 units, and bone-marrow compatible with A.H.A., on first admission to hospital (before treatment). *
After
D.T.T.
immunisation.
t During pertussis infection.