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The Significance of Eosinophil-counts
1042
commanding headlines in the newspapers repercussions of some political misfortune. Unhappily, this state of ignorance breeds apathy and
making a total of 80 mg. ; and the estimations are done just before the test and four hours after the last dose of A.C.T.H. The eosinophil-count in the four-hour test enables the more chauvinistic elements of the press is the quickest way of obtaining the desired answer, to attack W.H.O. on the ground that good money and it could even be used in the outpatient clinic. is being poured down an international drain with no PRUNTY gives figures to show that in the normal of return. In the total fact, however, hope adequate person there is a fall in the eosinophils of 50-98%, of for the is W.H.O. 1951 limited the average being 75% ; in Addison’s disease the expenditure year to 6,300,000 United States dollars (about i2 million) ; range was from + 12% to - 8% ; three patients the is we recall with Simmonds’s hypopituitarism gave z- 24%, - 4%, though comparison misleading, may that this represents about 1/200th part of the cost of and - 17%, and four with chromophobe adenoma of the National Health Service to Britain. Nobodv would the pituitary varied from - 15% to - 45%. These hold that the existence of an international organisation figures are thought to reflect the reduced adrenoabsolves member nations from their responsibility for cortical function in Addison’s and hypopituitary
are sure are
of
the
the health of their own citizens, and the bulk of expenditure on health must be a national matter. Nevertheless health has many vitally important international ramifications which can only be dealt with by international action. Moreover, the progress of civilisation depends on the willingness and ability of more highly developed countries to bring aid to backward communities. W.H.O. is now empowered to spend money on international health work at the rate of only about one farthing per head of world population per year ; and while expenditure remains at this level the world must be prepared to entertain only very modest hopes and expectations of what the organisation will achieve. All who can see beyond the boundaries of their own countries will be anxious to see it given a better chance to rise to the occasion.
diseases. Such a test as this, easy of application and economical in the scarce A.c.T.H., is designed for widespread application. But before it is put into practical use it would be as well. to know the normal variations in eosinophil-counts. Fortunately this point has been exhaustively examined by FiNiT Run3 of Oslo. Since eosinophils are relatively few it is important to have a proper counting technique. All forms of " dry the counting "-i.e., estimating eosinophil percentage of the total leucocyte-count by examining dry stained smears or coverslip preparations-have proved in many hands hopelessly inaccurate for a purpose such as this. RuD therefore turned to the " wet " tech. niques, in which the eosinophils are counted directly in a hsemocytometer. Here the difficulty is to prepare a dilution fluid which will enable the eosinophil cells to be counted easily and to be clearly distinguished of The EOSINOPHIL-COUNTS have come into prominence from other leucocytes, particularly neutrophil polyAfter experiment, RuD concluded that a lately because it has been found that administration morphs. solution containing magdala-red instead of eosin of pituitary adrenocorticotropic hormone (A.C.T.H.) the best staining, and he, like DUNGER,4 includes reduces the number of eosinophils in the peripheral gave acetone to destroy the red cells. He used a large blood. The A.C.T.H. acts by stimulating the adrenal counting-chamber (the Fuchs-Rosenthal) and an cortex to secrete corticosterone ; so, theoretically, the eosinopenia after giving A.C.T.H. could be used as ordinary white-cell pipette for giving a 1 : 20 dilution. Blood is obtained from a puncture, and according to 1 an indicator of adrenocortical function. We recently 5 the LuCEY finger and not the ear should be used; noted that a test on this basis has been proposed to detect patients who have poor adrenocortical reserves if citrated venous blood,is used, the count must be and would therefore be liable to surgical shock if made quickly because the eosinophils tend to gather into clumps not dispersed by subsequent submitted to and also shocked
Significance
Eosinophil-counts
operation,
patients
who are likely to benefit from treatment with extracts of adrenal cortex. An obvious extension of this use of A.C.T.H. is the estimation of adrenocortical function in patients with endocrine disorders, and this subject has been reviewed by PRUNTY.2 In a normal person, A.C.T.H. increases the excretion of 17-ketosteroids and 11-oxysteroids, and increases uric acid excretion in relation to creatinine excretion; but reduction in the eosinophil-count is far and away easier and quicker to estimate than either of these effects. PRUNTY also estimates the ratio of uric acid to creatinine excretion, which is within the competence of most hospital biochemical departments. He describes two proposed methods of conducting the The first is the " four-hour A.c.T.H. A.C.T.H. test. test " ; here a single intramuscular dose of 25 mg. of A.C.T.H. is given and estimations are carried out just before and four hours after the dose. In the " fortyeight hour A.C.T.H. test " 10 mg. of A.C.T.H. is given six-hourly, day and night, for forty-eight hours1. Leading article, Lancet, April 15, p. 719. 2. Prunty, F. T. G. J. clin. Path. 1950, 3, 87.
shaking.
The statistics of leucocyte counting in the haemocytometer have often been worked out, and, having regard to the relatively small number of cells that will be counted even over the whole area of the large chamber used, changes of less than about =i: 30% But RUD’S cannot be expected to be significant. of a elaborate statistical analysis large mass of in material shows that, fact, the eosinophil-count of a normal person varies through an even wider range in the 24 hours and from day to day. During the day the average eosinophil-count rises in the morning and afternoon to a maximum in the evening, and then falls
again; digestion, though traditionally blamed,
has
influence on the variation. Unfortunately individual counts vary greatly and may go up or down during the day. The following figures, taken from RUD’S table, show eosinophil-counts on normal persons taken at 10 A.M., and four hours later, at 2 P.M.
no
3. Rud, F. The Eosinophil-count in Health and Mental Disease. Oslo : Johan Grundt Tanum Forlag. 1947. 4. Dunger, R. Münch, med. Wschr. 1910, 57, 1942. 5. Lucey, H. C. J. clin. Path. 1950, 3, 146.
1043 Eosinophils Patient ]!,,Iale1 Male 2 Female7 Female 2
B
......
......
......
......
10 A.M. ]31 38 363 213
..
..
..
..
per 2 P.M. 56 755 106 350
c.mm.
..
..
..
..
Change -57% +99% 71% + 64%
It needs no elaborate calculation to see that these normal variations come within or exceed the range of changes regarded as of pathological significance. The conclusion to be drawn from all this is that a reasonable figure for a patient’s normal eosinophilcount cannot be obtained from a single count, or from several counts made at the same time. To minimise these spontaneous variations, RuD proposes that four counts should be made at fifteen-minute intervals in the afternoons of each of five or six successive days, and the average of these 20-24 counts will fairly represent the normal level. Even then the range of variation within the " normal " is large. For instance, he estimates 126 eosinophils per c.mm. as the mean count for absolutely normal persons. He then calculates that counts from 40 to 228 per c.mm. should be regarded as the normal range for males and 42-245 per c.mm. for females. In other words, at this level, reductions of less than 68% are not. significant, and normal increases of up to 80% must be reckoned with. The only answer to this sort of variation is to perform repeated tests with A.C.T.H. as RuD does for normals. If, indeed, any reliance at all is to be placed on eosinophil-counts, not only must technique be first-class but a programme of repeated four-hourly A.c.T.H. tests must be undertaken if any useful estimate of adrenocortical function is to be made from the counts. No-one familiar with the inescapable variations of cell counting will be surprised that only big changes in eosinophil-counts are likely to be significant. That they should be so large as RuD affirms is unexpected but not improbable. It is clear that his results must be taken seriously and carefully examined before diagnostic significance is attached to variations in the eosinophil-count.
Substitutes for Curare IN the last few years curare has been accepted as of the routine adjuncts to general anaesthesia for major surgery, first in Canadaand the U.S.A.,2 and then, as supplies improved, in Britain 3 and elsewhere. An inevitable result has been a search for cheaper, and if possible better, synthetic substitutes. The broad principles relating chemical structure to curare-like action were familiar to pharmacologists long before GRIFFITH drew the attention of anxsthetists to these muscle-relaxants, and many such substances had already been synthesised and investigated experimentally.4But the first synthetic substitute for curare to be given a wide clinical trial was ’Myanesin’ or mephenesin, introducedin 1947. This is a glycerol ether which produces relaxation of the skeletal muscles superficially resembling that of curare, but it acts at spinal-cord or basal-ganglia level, whereas curare neutralises the action of acetylcholine at the myoneural junctions ; the action of mephenesin is therefore not reversed by neostigmine, the recognised one
1. Griffith, H. R., Johnson, G. E. Anesthesiology, 1942, 3, 418. 2. Cullen, S. E. Surgery, 1943, 14, 2. 3. Mallinson, F. B. Lancet, 1945, ii, 75. 4. Ing, H. R. Physiol. Rev. 1936, 16, 527. 5. Berger, F. M., Bradley, W. Lancet, 1947, i, 97. Mallinson, F. B. Ibid, p. 98.
antidote to curare. Unfortunately, the commercial preparations of mephenesin sometimes produced h2emoglobinuria through intravascular haemolysis, and severe thrombophlebitis occasionally developed at the site of injection. After a good deal of controversy most anaesthetists have accepted the view that the risk of these side-effects is too serious for mephenesin to be used further in routine anaesthesia. Then last year two other synthetic substitutes
were
introduced-’ Flaxedil’ or tri-(&bgr;-diethylaminoethoxy) benzene triethiodide,6 and 010 or decamethonium iodide. Quantitative experiments on animals and on human volunteers have shown that flaxedil, in about five times the dosage, has an effect practically indistinguishable from that of d-tubocurarine and similarly reversed by neostigmine. So far the favourable reception of this drug by anaesthetists has been It has the advantage undisturbed by dissension. over curare of being readily miscible with thiopentone, so the two can be given in one syringe ; it is also cheaper and has not the same tendency to induce bleeding. In recent reports on its clinical use, DOUGHTYhas emphasised its value with thiopentone in preventing8 laryngeal spasm during intubation ; and RUDDELL has described its use with intravenous pethidine and gas-and-oxygen as approaching the ideal combination for major surgery. The position of C10 is less happy, mainly because no satisfactory antidote for it has yet been found. The first clinical report9 noted that theantidote was not neostigmine but pentamethonium iodide (C5), another member of the methonium series. And later experience has proved that neostigmine or physostigmine, instead of counteracting the effects of C10, may increase its relaxant action Both hexamethonium iodide and and (C5 pentamethonium C6) have a potent blocking action on autonomic ganglia, like that of tetraethylammonium bromide (T.E.A.B.). In anaesthetised patients they produce a fall in blood-pressure, which DAVISON 11 describes sharp " as profound and the rnanufacturers’ leaflet as precipitate " ; while HvctER, et al.12 refer to the state of affairs as " an alarming degree of circulatory collapse." DAVISON has made the interesting suggestion that this fall in blood-pressure after the injection of C5, which may amount to 75%, could be made use of in surgery to obtain a bloodless field. However, such an effect will certainly be undesirable in a patient already suffering from paralysis of the respiratory muscles. Apart from the difficulty over an antidote, the efficiency in anaesthesia of any newcomer to the group of relaxants must be measured with the well-tried d-tubocurarine as yardstick, and it is not yet clear that C10 possesses any advantages The muscular paralysis it over the natural drug. causes usually lasts a shorter time than that of curare, but occasionally, for reasons not yet understood, recovery may be delayed, and the delay seems to be a real hazard. GRAY 13 reports four such cases, in one of which muscular power did not return for "
6. Mushin, W. W., Wien, R., Mason, D. F. J., Langston, G. T. Ibid. 1949, i, 726. 7. Doughty, D. G. Ibid, May 13, p. 899. 8. Ruddell, J. S. Ibid. May 20, p. 953. 9. Organe, G., Paton, W. D. M., Zaimis, E. J. Ibid, 1949, i, 21. 10. Paton, W. D. M.. Zaimis, E. J. Brit. J. Pharmacol. 1949, 4, 381. 11. Davison, M. H. A. Lancet, Feb. 11, p. 252. 12. Hewer, A. J. H., Lucas, B. G. B., Prescott, F., Rowbotham, E. S. Ibid, 1949, i, 817. 13. Gray, A. J. Ibid, Feb. 11, p. 253.
commanding headlines in the newspapers repercussions of some political misfortune. Unhappily, this state of ignorance breeds apathy and
making a total of 80 mg. ; and the estimations are done just before the test and four hours after the last dose of A.C.T.H. The eosinophil-count in the four-hour test enables the more chauvinistic elements of the press is the quickest way of obtaining the desired answer, to attack W.H.O. on the ground that good money and it could even be used in the outpatient clinic. is being poured down an international drain with no PRUNTY gives figures to show that in the normal of return. In the total fact, however, hope adequate person there is a fall in the eosinophils of 50-98%, of for the is W.H.O. 1951 limited the average being 75% ; in Addison’s disease the expenditure year to 6,300,000 United States dollars (about i2 million) ; range was from + 12% to - 8% ; three patients the is we recall with Simmonds’s hypopituitarism gave z- 24%, - 4%, though comparison misleading, may that this represents about 1/200th part of the cost of and - 17%, and four with chromophobe adenoma of the National Health Service to Britain. Nobodv would the pituitary varied from - 15% to - 45%. These hold that the existence of an international organisation figures are thought to reflect the reduced adrenoabsolves member nations from their responsibility for cortical function in Addison’s and hypopituitary
are sure are
of
the
the health of their own citizens, and the bulk of expenditure on health must be a national matter. Nevertheless health has many vitally important international ramifications which can only be dealt with by international action. Moreover, the progress of civilisation depends on the willingness and ability of more highly developed countries to bring aid to backward communities. W.H.O. is now empowered to spend money on international health work at the rate of only about one farthing per head of world population per year ; and while expenditure remains at this level the world must be prepared to entertain only very modest hopes and expectations of what the organisation will achieve. All who can see beyond the boundaries of their own countries will be anxious to see it given a better chance to rise to the occasion.
diseases. Such a test as this, easy of application and economical in the scarce A.c.T.H., is designed for widespread application. But before it is put into practical use it would be as well. to know the normal variations in eosinophil-counts. Fortunately this point has been exhaustively examined by FiNiT Run3 of Oslo. Since eosinophils are relatively few it is important to have a proper counting technique. All forms of " dry the counting "-i.e., estimating eosinophil percentage of the total leucocyte-count by examining dry stained smears or coverslip preparations-have proved in many hands hopelessly inaccurate for a purpose such as this. RuD therefore turned to the " wet " tech. niques, in which the eosinophils are counted directly in a hsemocytometer. Here the difficulty is to prepare a dilution fluid which will enable the eosinophil cells to be counted easily and to be clearly distinguished of The EOSINOPHIL-COUNTS have come into prominence from other leucocytes, particularly neutrophil polyAfter experiment, RuD concluded that a lately because it has been found that administration morphs. solution containing magdala-red instead of eosin of pituitary adrenocorticotropic hormone (A.C.T.H.) the best staining, and he, like DUNGER,4 includes reduces the number of eosinophils in the peripheral gave acetone to destroy the red cells. He used a large blood. The A.C.T.H. acts by stimulating the adrenal counting-chamber (the Fuchs-Rosenthal) and an cortex to secrete corticosterone ; so, theoretically, the eosinopenia after giving A.C.T.H. could be used as ordinary white-cell pipette for giving a 1 : 20 dilution. Blood is obtained from a puncture, and according to 1 an indicator of adrenocortical function. We recently 5 the LuCEY finger and not the ear should be used; noted that a test on this basis has been proposed to detect patients who have poor adrenocortical reserves if citrated venous blood,is used, the count must be and would therefore be liable to surgical shock if made quickly because the eosinophils tend to gather into clumps not dispersed by subsequent submitted to and also shocked
Significance
Eosinophil-counts
operation,
patients
who are likely to benefit from treatment with extracts of adrenal cortex. An obvious extension of this use of A.C.T.H. is the estimation of adrenocortical function in patients with endocrine disorders, and this subject has been reviewed by PRUNTY.2 In a normal person, A.C.T.H. increases the excretion of 17-ketosteroids and 11-oxysteroids, and increases uric acid excretion in relation to creatinine excretion; but reduction in the eosinophil-count is far and away easier and quicker to estimate than either of these effects. PRUNTY also estimates the ratio of uric acid to creatinine excretion, which is within the competence of most hospital biochemical departments. He describes two proposed methods of conducting the The first is the " four-hour A.c.T.H. A.C.T.H. test. test " ; here a single intramuscular dose of 25 mg. of A.C.T.H. is given and estimations are carried out just before and four hours after the dose. In the " fortyeight hour A.C.T.H. test " 10 mg. of A.C.T.H. is given six-hourly, day and night, for forty-eight hours1. Leading article, Lancet, April 15, p. 719. 2. Prunty, F. T. G. J. clin. Path. 1950, 3, 87.
shaking.
The statistics of leucocyte counting in the haemocytometer have often been worked out, and, having regard to the relatively small number of cells that will be counted even over the whole area of the large chamber used, changes of less than about =i: 30% But RUD’S cannot be expected to be significant. of a elaborate statistical analysis large mass of in material shows that, fact, the eosinophil-count of a normal person varies through an even wider range in the 24 hours and from day to day. During the day the average eosinophil-count rises in the morning and afternoon to a maximum in the evening, and then falls
again; digestion, though traditionally blamed,
has
influence on the variation. Unfortunately individual counts vary greatly and may go up or down during the day. The following figures, taken from RUD’S table, show eosinophil-counts on normal persons taken at 10 A.M., and four hours later, at 2 P.M.
no
3. Rud, F. The Eosinophil-count in Health and Mental Disease. Oslo : Johan Grundt Tanum Forlag. 1947. 4. Dunger, R. Münch, med. Wschr. 1910, 57, 1942. 5. Lucey, H. C. J. clin. Path. 1950, 3, 146.
1043 Eosinophils Patient ]!,,Iale1 Male 2 Female7 Female 2
B
......
......
......
......
10 A.M. ]31 38 363 213
..
..
..
..
per 2 P.M. 56 755 106 350
c.mm.
..
..
..
..
Change -57% +99% 71% + 64%
It needs no elaborate calculation to see that these normal variations come within or exceed the range of changes regarded as of pathological significance. The conclusion to be drawn from all this is that a reasonable figure for a patient’s normal eosinophilcount cannot be obtained from a single count, or from several counts made at the same time. To minimise these spontaneous variations, RuD proposes that four counts should be made at fifteen-minute intervals in the afternoons of each of five or six successive days, and the average of these 20-24 counts will fairly represent the normal level. Even then the range of variation within the " normal " is large. For instance, he estimates 126 eosinophils per c.mm. as the mean count for absolutely normal persons. He then calculates that counts from 40 to 228 per c.mm. should be regarded as the normal range for males and 42-245 per c.mm. for females. In other words, at this level, reductions of less than 68% are not. significant, and normal increases of up to 80% must be reckoned with. The only answer to this sort of variation is to perform repeated tests with A.C.T.H. as RuD does for normals. If, indeed, any reliance at all is to be placed on eosinophil-counts, not only must technique be first-class but a programme of repeated four-hourly A.c.T.H. tests must be undertaken if any useful estimate of adrenocortical function is to be made from the counts. No-one familiar with the inescapable variations of cell counting will be surprised that only big changes in eosinophil-counts are likely to be significant. That they should be so large as RuD affirms is unexpected but not improbable. It is clear that his results must be taken seriously and carefully examined before diagnostic significance is attached to variations in the eosinophil-count.
Substitutes for Curare IN the last few years curare has been accepted as of the routine adjuncts to general anaesthesia for major surgery, first in Canadaand the U.S.A.,2 and then, as supplies improved, in Britain 3 and elsewhere. An inevitable result has been a search for cheaper, and if possible better, synthetic substitutes. The broad principles relating chemical structure to curare-like action were familiar to pharmacologists long before GRIFFITH drew the attention of anxsthetists to these muscle-relaxants, and many such substances had already been synthesised and investigated experimentally.4But the first synthetic substitute for curare to be given a wide clinical trial was ’Myanesin’ or mephenesin, introducedin 1947. This is a glycerol ether which produces relaxation of the skeletal muscles superficially resembling that of curare, but it acts at spinal-cord or basal-ganglia level, whereas curare neutralises the action of acetylcholine at the myoneural junctions ; the action of mephenesin is therefore not reversed by neostigmine, the recognised one
1. Griffith, H. R., Johnson, G. E. Anesthesiology, 1942, 3, 418. 2. Cullen, S. E. Surgery, 1943, 14, 2. 3. Mallinson, F. B. Lancet, 1945, ii, 75. 4. Ing, H. R. Physiol. Rev. 1936, 16, 527. 5. Berger, F. M., Bradley, W. Lancet, 1947, i, 97. Mallinson, F. B. Ibid, p. 98.
antidote to curare. Unfortunately, the commercial preparations of mephenesin sometimes produced h2emoglobinuria through intravascular haemolysis, and severe thrombophlebitis occasionally developed at the site of injection. After a good deal of controversy most anaesthetists have accepted the view that the risk of these side-effects is too serious for mephenesin to be used further in routine anaesthesia. Then last year two other synthetic substitutes
were
introduced-’ Flaxedil’ or tri-(&bgr;-diethylaminoethoxy) benzene triethiodide,6 and 010 or decamethonium iodide. Quantitative experiments on animals and on human volunteers have shown that flaxedil, in about five times the dosage, has an effect practically indistinguishable from that of d-tubocurarine and similarly reversed by neostigmine. So far the favourable reception of this drug by anaesthetists has been It has the advantage undisturbed by dissension. over curare of being readily miscible with thiopentone, so the two can be given in one syringe ; it is also cheaper and has not the same tendency to induce bleeding. In recent reports on its clinical use, DOUGHTYhas emphasised its value with thiopentone in preventing8 laryngeal spasm during intubation ; and RUDDELL has described its use with intravenous pethidine and gas-and-oxygen as approaching the ideal combination for major surgery. The position of C10 is less happy, mainly because no satisfactory antidote for it has yet been found. The first clinical report9 noted that theantidote was not neostigmine but pentamethonium iodide (C5), another member of the methonium series. And later experience has proved that neostigmine or physostigmine, instead of counteracting the effects of C10, may increase its relaxant action Both hexamethonium iodide and and (C5 pentamethonium C6) have a potent blocking action on autonomic ganglia, like that of tetraethylammonium bromide (T.E.A.B.). In anaesthetised patients they produce a fall in blood-pressure, which DAVISON 11 describes sharp " as profound and the rnanufacturers’ leaflet as precipitate " ; while HvctER, et al.12 refer to the state of affairs as " an alarming degree of circulatory collapse." DAVISON has made the interesting suggestion that this fall in blood-pressure after the injection of C5, which may amount to 75%, could be made use of in surgery to obtain a bloodless field. However, such an effect will certainly be undesirable in a patient already suffering from paralysis of the respiratory muscles. Apart from the difficulty over an antidote, the efficiency in anaesthesia of any newcomer to the group of relaxants must be measured with the well-tried d-tubocurarine as yardstick, and it is not yet clear that C10 possesses any advantages The muscular paralysis it over the natural drug. causes usually lasts a shorter time than that of curare, but occasionally, for reasons not yet understood, recovery may be delayed, and the delay seems to be a real hazard. GRAY 13 reports four such cases, in one of which muscular power did not return for "
6. Mushin, W. W., Wien, R., Mason, D. F. J., Langston, G. T. Ibid. 1949, i, 726. 7. Doughty, D. G. Ibid, May 13, p. 899. 8. Ruddell, J. S. Ibid. May 20, p. 953. 9. Organe, G., Paton, W. D. M., Zaimis, E. J. Ibid, 1949, i, 21. 10. Paton, W. D. M.. Zaimis, E. J. Brit. J. Pharmacol. 1949, 4, 381. 11. Davison, M. H. A. Lancet, Feb. 11, p. 252. 12. Hewer, A. J. H., Lucas, B. G. B., Prescott, F., Rowbotham, E. S. Ibid, 1949, i, 817. 13. Gray, A. J. Ibid, Feb. 11, p. 253.