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The Application and Interpretation of the Blood Sedimentation Test in Clinical Medicine
Medical Clinics of North America September, 1937. Baltimore Number
CLINIC OF DR. MAXWELL M. WINTROBE THE JOHNS HOPKINS HOSPITAL
THE APPLICATION AND INTERPRETATION OF THE BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
IN little more than a decade the blood sedimentation test has attained widespread use. For this there are two reasons. One is the simplicity of the test; the other is its applicability to all phases of medicine. The principle of the test is extremely simple. The blood is essentially a suspension of corpuscles in plasma. For the sedimentation test, blood is mixed with an anticoagulant, and the stability of the suspension is measured by putting some blood into a narrow tube which stands in a vertical position. The sedimentation of the red corpuscles can then be observed. Although Fahraeus, a Swede, is responsible for the introduction of the sedimentation test into modern medicine, it is in a sense as old as the theory of the four humors. When blood was withdrawn from a healthy person, it was observed that it clotted and formed two portions, the serum and the clot. On the other hand, when blood was withdrawn from a diseased person, sedimentation being more rapid, separation of several layers occurred before clotting permitted no further change to take place. In this blood, four portions could be distinguished: (1) the uppermost yellowish fluid, formed by the blood serum ("cholera" or "yellow bile"); (2) a grayishwhite layer of fibrin in the upper portion of the clot ("phlegma" or "mucus"); (3) a bright red layer made up chiefly of red corpuscles ("sanguis"); and (4) a dark red, almost black portion made up of red corpuscles deprived of oxygen ("melancholia" or "black bile"). In the medical philosophy of the ancient Greeks, ill health was thought to be due to the failure VOL. 21-97
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MAXWELL M. WINTROBE
of these four "humors" to mix. In the medical thought of later years the phlegm a received chief attention and as the "crusta inflammatoria," "buffy coat" or "size" was thought to be the cause of disease. It was to rid the body of this supposedly harmful substance that venesection was performed. Fahraeus' discovery of the value of the sedimentation test was quite accidental. In his search for an early test for pregnancy he found that the rate of sedimentation of the ,red corpuscles is increased not only in pregnancy but also in many diseases. THE NATURE OF THE SEDIMENTATION PHENOMENON
Little is known about the true nature of the sedimentation phenomenon. Fahraeus pointed out that the suspension stability of the blood is determined by the radius of the suspended particles. Variations in the latter depend on differences in the degree of aggregation of the red corpuscles, with resulting formation of larger or smaller particles. In bloods in which sedimentation is rapid large aggregates are formed. The cause of the increased aggregation or rouleaux formation of the corpuscles is not known. It appears to be some factor present chiefly in the plasma, for sedimentation is more rapid in plasma than in serum. There is a close correlation between the quantity of plasma fibrinogen and sedimentation rate, and there is some correlation between the amount of plasma globulin and the rate of sedimentation. Yet it is not correct to assume, as many writers have done, that the increased rate is therefore due to increases in plasma fibrinogen or globulin. The real causative factor has not yet been demonstrated. METHODS FOR DETERMINING SEDIMENTATION RATE
Numerous methods have been .devised for determination of sedimentation rate. Actually these are all modifications of two principal methods. The blood, to which an anticoagulant has been added, is placed in a narrow tube which is fixed in a vertical position. Then either the time required for the upper level of sedimenting corpuscles to fall a specified distance is measured (Linzenmeyer method), or the distance the cornusdes fall in a specified interval of time is noted (Westergren
BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
1539
method) . The latter method is more generally used because it is so simple to fill the sedimentation tube and then to pay no further attention to it until the time chosen to read it has elapsed. Some investigators recommend that readings be made at short, usually five-minute, intervals. When this is done and the readings are recorded on a chart, every variation from Sea.
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Fig. 107.-Sedimentation curves in the normal and in various disorders: A, normal; B, mild tonsillitis; C, mitral insufficiency without active rheumatic infection; D, chronic bilateral salpingitis and endocervicitis; E, bronchitis and bronchiectasis; F, acute pharyngitis, laryngitis and bronchitis. Both the "corrected" and "uncorrected" sedimentation rates are recorded in the last column.
an almost horizontal line to one which quickly falls in a vertical direction, may be encountered (Fig. 107). It can then be observed that in the process of sedimentation there are three phases; namely: ( 1) a preliminary period during which aggregation of the red corpuscles takes place, (2) ~ period of rapid fall, and finally (3) the phase of packing when the corpuscular masses, piling on one another, slow up the rate of sedimentation.
MAXWELL M. WINTROBE
It is the phase of rapid fall which is thought to be of chief significance in the sedimentation test and for this reason charting of the changes which occur at five- or -ten-minute intervals has been recommended. Actually, however, the distance which the corpuscles have fallen at the end of one hour is accounted for chiefly by this phase of rapid fall. Consequently, in the opinion of most workers, it is quite adequate to make only one reading, at the end of one hour. By doing this, the simplicity of the sedimentation test is preserved and yet adequate information is gained. In the interpretation of the sedimentation test it is important to make certain that the test has been properly carried out, because a number of simple variations from the standard technic may cause delay or increase in the rate of fall of the corpuscles. Thus, if an excess of anticoagulant is used, the rate may be delayed. If the tube is allowed to stand even at an angle of 87 degrees rather than 90 degrees, the speed of sedimentation is markedly accelerated. It is also important to use tubes of uniform Qore and length. Again, blood should be used for the sedimentation test within four hours qf its withdrawal from the patient, as otherwise the rate may be less than that in freshly drawn blood. Furthermore, excessive cold will slow sedimentation while heat causes acceleration. In order to ensure uniformity of results, we follow a standardized technicl l using the hematocrit devised by the writer as the sedimentation tube (Fig. 108). "1. Five cc. of venous blood is collected -by means of a dry syringe and needle and mixed in a small bottle containing 4 mg. solid potassium oxalate and 6 mg. solid ammonium oxalate. This concentration of oxalate does not alter the sedimentation rate as compared with that of blood collected in heparin. Less than 1 cc. of blQod is needed for the sedimentation test. The remainder can be used for other blood examinations. • "2. The blood so collected 'should be used for the determination of sedimentation rate within four hours of its time of collection. Further delay may be associated with increased suspension stability of the blood. _-. "l... The hematocrit is filled with blood to the 10 cm. mark.
BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
I54I
The upper level of sedimenting corpuscles may be read at frequent intervals, or more simply, a single reading may be made at the end of one hour. "4. Since sedimentation rate increases with increasing temperature, the sedimentation test should be carried out at a temperature not less than 22° nor greater than 27° C. 'Within this range variations reSUlting from differences in tem-
Fig. l08 .-Sedimentation of blood in hematocrit. A, Sedimentation of the blood of a normal male adult at the end of one hour; B, sedimentation of the blood of a normal female; C, the same sample of blood as in B, fol1o~ing centrifugation to secure complete packing of corpuscles; D, E and Frepresent the blood of 3 patients with slight, moderate, and marked increases in sedimentation velocity. (Wintrobe, International Clin., J. B. Lippincott Co., 1936, vol. II, 46th series, p. 34.)
perature are small. If the blood used has previously been kept in a refrigerator it should first be permitted to attain the above temperature before being used. "5. The hematocrit should be kept in an exact vertical position 'during the sedi~entation of the blood corpuscles, for when the instrument stands at an angle of even 3 ° from the vertical, significant acceleration of sedimentation takes place. "6. After sedimentation rate has been determined, the
MAXWELL M. WINTROBE
hematocrit containing the blood should be centrifugalized and volume of packed red cells determined. The sedimentation rate may then be corrected for alterations due to anemia.". It has been repeatedly shown tha1 variations in the quantity of sedimenting red corpuscles cause differences in sedimen-
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Fig. 109.-The logarithmic CUrve on which this chart is based is heavily outlined. The mean normal volume of packed red cells for men (47 cc.) and for women (42 cc.) are also heavily outlined and the range of normal sedimentation is represented by solid and open columns for each sex, respectively .. For correcting sedimentation rate, find on the chart the horizontal line corresponding to the sedimentation rate for the patient; find also the vertical line corresponding to the volume of packed red cells in the patient's blood. Select the curve lying nearest to the .point of junction of the horizontal and the vertical line and follow this curve to the normal line for the sex of. the pati~nt.. The horizontal line corresponding to this last. point of juncture· gives the corrected sedimentation rate. A simpler method of c.orrection is to correct all values to the volume of packed red cells normal for the male (47 cc.). Thus a single standard of normal can be used for both sexes. See text. (Wintrobe and Landsberg in Amer. Jour. Med.Sei., 189: 102 [Jan.], 1935.)
tation rate. A decrease in their number, as in anemia, per.mits a more rapid rate of fall, whereas "When they are increased above the normal, practically no sedimentation occurs, -Suc1ialterations in sedimentation rate due to differences in the
BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
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quantity of corpuscles may be allowed for by the use of the chart shown in Fig. 109. There are several reasons why the hematocrit should be used for the sedimentation test. The chief one, is that the quantity of corpuscles may easily be determined in the same tube by simply centrifugalizing (3000 revolutions per minute, for thirty minutes) after sedimentation has been recorded. As a routine procedure this is useful not only in simplifying the correction of sedimentation rate, but it is a quick and accurate means of discovering whether the patient has anemia or polycythemia.. Furthermore, from the thickness of the layer of leukQcytes and platelets which is found above the layer of packed red corpuscles, one may gauge roughly the quantity of these corpuscles in the blood. Finally, the icterus index may be measured by comparing the color of the plasma in the hematocrit with the color or a series of tubes containing potassium dichromate solution in various dilutions. 9 In infants and young children, when venipuncture is not practical, a modification of the above method may be employed. The heel or finger is punctured deeply enough to secure a free flow of blood.. With a capillary pipet blood is transferred quickly in successive small amounts to a small test tube containing approximately one-tenth the amount of oxalate mixture used for 5 cc. venous blood. Thorough mixing of blood and anticoagulant must be secured. After a little more than 0;5 cc. of blood has been obtained, it is transferred to the hematocrit, which is filled only half way. Sedimentation is then recorded in the usual manner, following which the blood is centrifugalized. To obtain the volume of packed red cells per 100 cc. of blood, the reading must, of course, be multiplied by two. NORMAL SEDIMENTATION RATE.
PHYSIOLOGIC VARIATIONS
The normal sedimentation rate differs according to the method used. It is therefore essential that a standard pro"' cedure be followed and the normal for the method employed be known. In health a slight interindividual variation in sedimenta~ tion rate occurs, and there is also a distinct difference in rate between the sexes. Thus, when the technic described was em-
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MAXWELL M. WINTROBE
ployed, the sedimentation rate in healthy men averaged 3.7 mm. at the end of one hour (0 to 6.5 mm. in 86 per cent of the subjects) while in healthy women it averaged 9.6 mm. (0 to 15 mm. in 86 per cent). This difference, however, is largely accounted for by the quantitative difference in corpuscles in the blood of men and women. When these sedimentation rates in 136 men and in 100 women were "corrected" to a volume of packed red cells of 47 cc. per 100 cc. of blood, the sedimentation rate in 72 per cent of the men and women ranged between 0 and 6 mm. at the end of one hour, and in an additional 16 per cent, it was 7 to 10 mm. These values may be considered the limits of normal. As has already been mentioned, sedimentation is accelerated during pregnancy. From about the tenth or twelfth week there is usually a gradual increase in rate which does not return to normal until the third or fourth week postpartum. A slight fluctuation of the rate of sedimentation occurs in relation to the menstrual cycle, but the changes are so small that, from a clinical standpoint, they are of no significance. Other factors, such as the ingestion of food, or. short violent exercise, are of no importance. VARIATIONS IN SEDIMENTATION RATE IN DISEASE
Methods for the determination of sedimentation rates and the variations under physiologic conditions have been discussed in some detail because, unless the technician performing the test and the physician interpreting the results are fully cognizant of these details, important errors may be introduced and the results in consequence become misleading. When the method is understood, however, the test becomes an extremely valuable aid in almost all phases of clinical medicine. In the interpretation of the sedimentation test it is essential to bear in mind that it is a nonspecific reaction which may be compared with the temperature chart, the pulse rate and the leukocyte count, in that it gives information of a general character. It is in fact less specific than the leukocyte count when the differential examination of the white corpuscles is included under the latter head, for then the leukocyte count may offer a clue to the nature of the disease . . ,.Itway be asked what purpose there is in the sedimentation
BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
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test if it affords information of the same type as the temperature ·and pulse record and the leukocyte count. In answer it may be pointed out that in many disorders and even in certain stages of febrile diseases, there may be no fever or tachycardia and the leukocyte and differential counts may be essentially normal; yet in many of these instances the sedimentation rate may be increased. The sedimentation test is, thus, complementary to these valuable clinical tools. Like them it is a measure of the presence and intensity of morbid processes within the body. Sometimes an increased sedimentation rate is found and nothing can be discovered to explain this increase. So little is still understood about the exact nature of the sedimentation reaction that a complete definition of the circumstances under which alterations in sedimentation rate may be expected to occur cannot be given. Yet, if the sedimentation rate in any patient is found repeatedly to be increased, the careful physician will seek to discover the cause. The sedimentation rate is increased in all acute general infections. The degree of increase tends to parallel the severity of the infection. Although an accelerated rate generally does not appear as soon as elevation of temperature, the abnormal rate subsides more slowly and for this reason it may serve as a guide to the condition of the morbid process after fever and tachycardia have disappeared. When complications develop, the sedimentation rate is further increased and this fact may serve to indicate the onset of a complication before its nature and site have been determined. In localized acute inflammatory conditions, variations in sedimentation rate depend on the nature and severity of the morbid process. In simple catarrhal inflammation, such as acute catarrhal appendicitis, simple rhinitis and bronchitis, the sedimentation rate tends to be normal whereas in localized acute suppurations, such as pelvic inflammatory disease, suppurative mastoiditis or sinusitis, there is a pronounced acceleration of sedimentation. In chronic localized infections the rate varies with the extent and nature of the infection, normal values often being found in chronic tonsillitis, whereas increased rates occur in chronic bronchitis, tuberculosis, syphilis, nephritis and liver abscess. Generally speaking, sedimentation rate tends to be normal in
MAXWELL M. WINTROBE
uninfected benign new growths whereas it may be increased when a malignant tumor is present. The latter, however, is not necessarily the case and the factors which probably determine whether the sedimentation rate is accelerated or not include the anatomic character of the growth, its vascularity, the tendency to break down, the degree of resorption of tumor tissue, the degree of reactive inflammation, and the location as well as the presence of metastases. 4 SEDIMENTATION RATES IN VARIOUS DISORDERS
The sedimentation reaction has been demonstrated to be of particular value in following the course of a number of disorders. It will be of interest to discuss these in detail for they illustrate the peculiar advantages of this test. That the sedimentation test is extremely valuable in phthisiology is attested by numerous reports. In pulmonary tuberculosis, variations in sedimentation rate reflect the intensity of the morbid process more accurately than the pulse, temperature, weight, sputum, symptoms. or physical signs. At the Trudeau Sanatorium" it was found that in some instances an increase in rate gave warning of relapses even before new shadows were found in the roentgenogram. No better claim for the test than this can be made, for the value of the roentgenogram as an index of the condition of the tuberculous patient is well established. When one considers the expensiveness of roentgenography and the difficulties sometimes encountered in interpretation, as compared with the simplicity of the sedimentation test, the importance of the latter becomes apparent. It does not, of course, offer any evidence regarding the extensiveness of the tuberculous lesion. The sedimentation reaction indicates the intensity and activity of the inflammatory process. In extensive and yet quiescent fibrotic lesions, sedimentation is less than in limited tuberculous processes with marked tissue disintegration. The sedimentation test is ravored by some investigators even in comparison with the various leukocytic indices. The value of the latter depends in large measure on the ability and interest of the observer. Differential leukocyte counts perfornred in a cursory manner by persons with inadequate train-
BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
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ing are of little value. Here again the simplicity of the sedimentation test gives it an important advantage. In patients who are not under constant observation, the sedimentation test is especially valuable when repeated at regular intervals. Again, it is important in those receiving pneumothorax therapy for here collapse of the lung makes physical signs and roentgenograms of less value than usual. A few observers have reported the finding of normal sedimentation rates in the presence of active tuberculous infectidn. Most workers will agree, however, that such a finding is extremely rare and usually represents a technical error. At the same time it must be pointed out that in severely cachectic stages of tuberculosis, the sedimentation rate has occasionally been found to be normal, or at least slower than earlier in the disease. In the study of rheumatic fever, the sedimentation test is gaining an important place. Its value is found particularly in rheumatic carditis, where it is regularly accelerated. Except when associated with congestive heart failure, a decreasing rate nearly always reflects diminishing activity of the rheumatic process and as an index of this the sedimentation reaction is much more valuable than the temperature, pulse rate or leukocyte count. It has been found that an increasing rate presages clinical exacerbation and many clinicians now permit no activity on the part of the patient until the sedimentation rate has returned to normal. . In acute cardiac infarction and in syphilitic aortitis the sedimentation rate is accelerated, whereas in angina pectoris and hypertensive heart disease the rate is normal. In cases of cardiac infarction the test is a valuable guide in deciding upon the duration of rest because in this condition, as in rheumatic carditis, an accelerated rate may be the only evidence of activity of the morbid process. Rheumatoid, tuberculous and gonorrheal arthritis, and other inflammatory diseases of bones and joints are associated with accelerated sedimentation rates, whereas in hypertrophic or osteo-arthritis, the rate is usually normal. For this reason, the test has been used in aiding the differentiation of rheumatoid from osteo-arthritis. In this respect, however, it has not always been found reliable.
MAXWELL M. WINTROBE
In gynecologic practice, the sedimentation test has achieved a position of great usefulness. The principles involving its use are the same as those already outlined. Uncomplicated benign tumors, such as fibromyomata or ovarian cysts, mild infections or quiescent inflammatory disease, are associated with no increase in sedimentation rate, whereas when there is active inflammatory disease the rate is accelerated. Degeneration of benign tumors, twisting of the pedicle of an ovarian cyst, and often malignant disease, are associated with rapid rates. Since the sedimentation rate is not usually increased in pregnancy until the tenth or twelfth week, the test can be used as an aid in differential diagnosis and as an indication of the presence of complications in cases of abortion and ectopic pregnancy. Retention of uninfected products of conception, hemorrhage or infection causes acceleration of various degrees. In acute and subacute salpingitis the rate is greatly increased whereas in acute appendicitis only slight increases usually occur. Subsidence of pelvic infection is associated with return of sedimentation rate toward normal. Many gynecologists have found this fact a useful guide in indicating a favorable time for elective operations. In aiding the study of gastro-intestinal disorders, the sedimentation test has not as yet attained the general application that it enjoys in the fields already discussed. It has been used by some clinicians, however, to facilitate the differentiation of functional from organic disturbances, benign ulceration from malignant, simple diarrhea from enteritis and colitis. The usefulness of the test in these respects is limited. As has already been pointed out, an increased sedimentation rate is not always associated with malignancy and whether or not the rate is accelerated in inflammatory disorders depends on the severity of the inflammatory process. It is of interest to note that in parenchymatous dis,eases of the liver, sedimentation rate may be actually less than normal. This has been explained as the result of failure to form fibrinogen which, as already mentioned, is closely related to the sedimentation reaction. Undoubtedly one of the most important uses of the sedimentation test is in calling attention to the presence of more or l~s occult diseases. As a routine procedure to be performed
BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
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as part of the general examination of a patient, the test is very valuable, for an accelerated rate may not infrequently be the sole evidence of the presence of disease and serves in this way as an indication of the necessity for further study of the patient. When the sedimentation test is carried out in the manner here described and the other steps in the use of the hematocrit performed, one obtains, as the result of a minimal expenditure of time and effort, information regarding not only sedimentation rate but also concerning the presence or not of anemia, leukocytosis and icterus. When the sedimentation test is used as a routine proc~dure in the examination of a patient, it must not be assumed that a normal rate necessarily signifies the absence of disease .. Occasionally it is found, although it is very rare, that the rate is normal in spite of the presence of organic disease. Thus among 444 cases at the Diagnostic Clinic of the Johns Hopkins Hospital in which the corrected sedimentation rate was 10 mm. or less, manifest organic disease was found in 8 instances. In· one case of continued fever of undetermined origin the sedimentation was 0 at the end of one hour. In two cases of chronic nephritis with marked renal insufficiency the rate was o and 1 mm., respectively, while in a patient who had active pulmonary tuberculosiS' as well as hay fever, it was 7 mm. Again, in a patient convalescing from amebiasis who still had some elevation of temperature, the rate was 8 mm. Three of these 8 cases illustrated the importance of recording the uncorrected as well as the corrected sedimentation rate. One patient had acute pyelitis with hydronephrosis, another had carcinoma of the descending colon and the third had cystitis, pyelitis and renal insufficiency. The corrected rates were practically normal (8, 9 and 10 mm., respectively), whereas the uncorrected values were markedly accelerated (37, 30· and 39 mm., respectively). In these instances the accelerated rates were masked by the correction for anemia. A study at the Henry Phipps Institute in Philadelphia 2 stresses the importance of paying due regard to sedimentation rates which are found to be accelerated although the general examination of the patient is negative. In 177 of 328 individuals who, after routine examination, had been considered
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MAXWELL M. WINTROBE
healthy or suffering from only trivial ailments, the sedimentation rate was increased. Reexamination of these patients revealed basal nontuberculous infiltration of the lungs in 96, "latent" apical tuberculosis in 32, positive Wassermann reactions in 35, pelvic inflammatory disease in 5, and possible carcinoma of the lung in 1. Only in 8 cases was there no abnormality found on reexamination. Criticism of the sedimentation test arises from a failure to understand the nature of the reaction and, as with all laboratory procedures, from failure to appreciate the importance of attention to what may appear to be trivial technical details. It must be emphasized that slight alterations from the standard technic may cause great differences in results. As has been pointed out, "correction" for anemia may occasionally be misleading. While correction should be made, the uncorrected rate and the hematocrit reading should be recorded as welI' as the corrected sedimentation rate. Again, it may be repeated that normal sedimentation velocity may occasionally be found in the presence of disease. Finally, it must be stressed that the reaction is a nonspecific one referable to QO particular disease and only indirectly of value in differential diagnosis. SELECTED RECENT REFERENCES AND REVIEWS 1. Coburn, A. F., and Kapp, E. M.: Observations on the Development of the High Blood Sedimentation Rate in Rheumatic Carditis, Jour. Clin. Invest., 15: 715, 1936. 2. Cutler, J. W.: The Practical Application of the Blood Sedimentation Test in General Medicine, Amer. Jour. Med. Sci., 183: 643, 1932. 3. Fahraeus, R.: The Suspension Stability of the Blood, Acta med. Scandinav., 56: 1, 1921. 4. Katz, G., and Leffkowitz, M.: Die Blutkorperchensenkung, Ergeb. d. inn. Med. u. Kinderh~ilk., 33: 266, 1928 (Bibliography). 5. Kelley, W. 0.: The Erythrocyte Sedimentation Rate in Estimating Activity in Pulmonary Tuberculosis, Amer. Rev. Tuberc., 34: 489, 1936. 6. Lucia, S. P., Blumberg, T., Brown, J. W., and Gospe, S. M.: The Relation Between the Suspension Stability of Erythrocytes and Various Constituents of Pathologic Human Blood, Amer. Jour. Med. Sci., 192: 179, 1936. 7. Shookhoff, C., Douglas, A. H., and Rabinowitz, A.: Sedimentation Time in Acute Cardiac Infarction, Ann. Int. Med., 9: 1101, 1936. 8. Smith, C. H.: A Method for Determining the Sedimentation Rate and Red C,ell Volume in Infants and Children with the Use of Capillary Blood. Ariler."Jour. Med. Sci., 192: 73, 1936.
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9. Wintrobe, M. M.: Macroscopic Examination of the Blood, Amer. Jour. Med. Sci., 186: 58, 1933. 10. Wintrobc, M. M.: The Erythrocyte Sedimentation Test, Internat. Clinics, J. B. Lippincott Co., Phila., 46th series, 2: 34 (June), 1936 (Bibliography) . 11. Wintrobe, M. M., and Landsbcrg, J. W.: A Standardized Technique for the Blood Sedimentation Test, Amer. Jour. Med. Sci., 189: 102, 1935. 12. Wood, P.: The Erythrocyte Sedimentation Rate in Diseases of the Heart, Quart. Jour. Med., 5: 1, 1936.
CLINIC OF DR. MAXWELL M. WINTROBE THE JOHNS HOPKINS HOSPITAL
THE APPLICATION AND INTERPRETATION OF THE BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
IN little more than a decade the blood sedimentation test has attained widespread use. For this there are two reasons. One is the simplicity of the test; the other is its applicability to all phases of medicine. The principle of the test is extremely simple. The blood is essentially a suspension of corpuscles in plasma. For the sedimentation test, blood is mixed with an anticoagulant, and the stability of the suspension is measured by putting some blood into a narrow tube which stands in a vertical position. The sedimentation of the red corpuscles can then be observed. Although Fahraeus, a Swede, is responsible for the introduction of the sedimentation test into modern medicine, it is in a sense as old as the theory of the four humors. When blood was withdrawn from a healthy person, it was observed that it clotted and formed two portions, the serum and the clot. On the other hand, when blood was withdrawn from a diseased person, sedimentation being more rapid, separation of several layers occurred before clotting permitted no further change to take place. In this blood, four portions could be distinguished: (1) the uppermost yellowish fluid, formed by the blood serum ("cholera" or "yellow bile"); (2) a grayishwhite layer of fibrin in the upper portion of the clot ("phlegma" or "mucus"); (3) a bright red layer made up chiefly of red corpuscles ("sanguis"); and (4) a dark red, almost black portion made up of red corpuscles deprived of oxygen ("melancholia" or "black bile"). In the medical philosophy of the ancient Greeks, ill health was thought to be due to the failure VOL. 21-97
1537
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of these four "humors" to mix. In the medical thought of later years the phlegm a received chief attention and as the "crusta inflammatoria," "buffy coat" or "size" was thought to be the cause of disease. It was to rid the body of this supposedly harmful substance that venesection was performed. Fahraeus' discovery of the value of the sedimentation test was quite accidental. In his search for an early test for pregnancy he found that the rate of sedimentation of the ,red corpuscles is increased not only in pregnancy but also in many diseases. THE NATURE OF THE SEDIMENTATION PHENOMENON
Little is known about the true nature of the sedimentation phenomenon. Fahraeus pointed out that the suspension stability of the blood is determined by the radius of the suspended particles. Variations in the latter depend on differences in the degree of aggregation of the red corpuscles, with resulting formation of larger or smaller particles. In bloods in which sedimentation is rapid large aggregates are formed. The cause of the increased aggregation or rouleaux formation of the corpuscles is not known. It appears to be some factor present chiefly in the plasma, for sedimentation is more rapid in plasma than in serum. There is a close correlation between the quantity of plasma fibrinogen and sedimentation rate, and there is some correlation between the amount of plasma globulin and the rate of sedimentation. Yet it is not correct to assume, as many writers have done, that the increased rate is therefore due to increases in plasma fibrinogen or globulin. The real causative factor has not yet been demonstrated. METHODS FOR DETERMINING SEDIMENTATION RATE
Numerous methods have been .devised for determination of sedimentation rate. Actually these are all modifications of two principal methods. The blood, to which an anticoagulant has been added, is placed in a narrow tube which is fixed in a vertical position. Then either the time required for the upper level of sedimenting corpuscles to fall a specified distance is measured (Linzenmeyer method), or the distance the cornusdes fall in a specified interval of time is noted (Westergren
BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
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method) . The latter method is more generally used because it is so simple to fill the sedimentation tube and then to pay no further attention to it until the time chosen to read it has elapsed. Some investigators recommend that readings be made at short, usually five-minute, intervals. When this is done and the readings are recorded on a chart, every variation from Sea.
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Fig. 107.-Sedimentation curves in the normal and in various disorders: A, normal; B, mild tonsillitis; C, mitral insufficiency without active rheumatic infection; D, chronic bilateral salpingitis and endocervicitis; E, bronchitis and bronchiectasis; F, acute pharyngitis, laryngitis and bronchitis. Both the "corrected" and "uncorrected" sedimentation rates are recorded in the last column.
an almost horizontal line to one which quickly falls in a vertical direction, may be encountered (Fig. 107). It can then be observed that in the process of sedimentation there are three phases; namely: ( 1) a preliminary period during which aggregation of the red corpuscles takes place, (2) ~ period of rapid fall, and finally (3) the phase of packing when the corpuscular masses, piling on one another, slow up the rate of sedimentation.
MAXWELL M. WINTROBE
It is the phase of rapid fall which is thought to be of chief significance in the sedimentation test and for this reason charting of the changes which occur at five- or -ten-minute intervals has been recommended. Actually, however, the distance which the corpuscles have fallen at the end of one hour is accounted for chiefly by this phase of rapid fall. Consequently, in the opinion of most workers, it is quite adequate to make only one reading, at the end of one hour. By doing this, the simplicity of the sedimentation test is preserved and yet adequate information is gained. In the interpretation of the sedimentation test it is important to make certain that the test has been properly carried out, because a number of simple variations from the standard technic may cause delay or increase in the rate of fall of the corpuscles. Thus, if an excess of anticoagulant is used, the rate may be delayed. If the tube is allowed to stand even at an angle of 87 degrees rather than 90 degrees, the speed of sedimentation is markedly accelerated. It is also important to use tubes of uniform Qore and length. Again, blood should be used for the sedimentation test within four hours qf its withdrawal from the patient, as otherwise the rate may be less than that in freshly drawn blood. Furthermore, excessive cold will slow sedimentation while heat causes acceleration. In order to ensure uniformity of results, we follow a standardized technicl l using the hematocrit devised by the writer as the sedimentation tube (Fig. 108). "1. Five cc. of venous blood is collected -by means of a dry syringe and needle and mixed in a small bottle containing 4 mg. solid potassium oxalate and 6 mg. solid ammonium oxalate. This concentration of oxalate does not alter the sedimentation rate as compared with that of blood collected in heparin. Less than 1 cc. of blQod is needed for the sedimentation test. The remainder can be used for other blood examinations. • "2. The blood so collected 'should be used for the determination of sedimentation rate within four hours of its time of collection. Further delay may be associated with increased suspension stability of the blood. _-. "l... The hematocrit is filled with blood to the 10 cm. mark.
BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
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The upper level of sedimenting corpuscles may be read at frequent intervals, or more simply, a single reading may be made at the end of one hour. "4. Since sedimentation rate increases with increasing temperature, the sedimentation test should be carried out at a temperature not less than 22° nor greater than 27° C. 'Within this range variations reSUlting from differences in tem-
Fig. l08 .-Sedimentation of blood in hematocrit. A, Sedimentation of the blood of a normal male adult at the end of one hour; B, sedimentation of the blood of a normal female; C, the same sample of blood as in B, fol1o~ing centrifugation to secure complete packing of corpuscles; D, E and Frepresent the blood of 3 patients with slight, moderate, and marked increases in sedimentation velocity. (Wintrobe, International Clin., J. B. Lippincott Co., 1936, vol. II, 46th series, p. 34.)
perature are small. If the blood used has previously been kept in a refrigerator it should first be permitted to attain the above temperature before being used. "5. The hematocrit should be kept in an exact vertical position 'during the sedi~entation of the blood corpuscles, for when the instrument stands at an angle of even 3 ° from the vertical, significant acceleration of sedimentation takes place. "6. After sedimentation rate has been determined, the
MAXWELL M. WINTROBE
hematocrit containing the blood should be centrifugalized and volume of packed red cells determined. The sedimentation rate may then be corrected for alterations due to anemia.". It has been repeatedly shown tha1 variations in the quantity of sedimenting red corpuscles cause differences in sedimen-
IU
Z
0 70
; j
~60
w I-
~ 50 .
Z
040
I-
<
0
I30 Z . w
:::2
is
0
20
w
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o
5
10
0
25
30
35
Fig. 109.-The logarithmic CUrve on which this chart is based is heavily outlined. The mean normal volume of packed red cells for men (47 cc.) and for women (42 cc.) are also heavily outlined and the range of normal sedimentation is represented by solid and open columns for each sex, respectively .. For correcting sedimentation rate, find on the chart the horizontal line corresponding to the sedimentation rate for the patient; find also the vertical line corresponding to the volume of packed red cells in the patient's blood. Select the curve lying nearest to the .point of junction of the horizontal and the vertical line and follow this curve to the normal line for the sex of. the pati~nt.. The horizontal line corresponding to this last. point of juncture· gives the corrected sedimentation rate. A simpler method of c.orrection is to correct all values to the volume of packed red cells normal for the male (47 cc.). Thus a single standard of normal can be used for both sexes. See text. (Wintrobe and Landsberg in Amer. Jour. Med.Sei., 189: 102 [Jan.], 1935.)
tation rate. A decrease in their number, as in anemia, per.mits a more rapid rate of fall, whereas "When they are increased above the normal, practically no sedimentation occurs, -Suc1ialterations in sedimentation rate due to differences in the
BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
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quantity of corpuscles may be allowed for by the use of the chart shown in Fig. 109. There are several reasons why the hematocrit should be used for the sedimentation test. The chief one, is that the quantity of corpuscles may easily be determined in the same tube by simply centrifugalizing (3000 revolutions per minute, for thirty minutes) after sedimentation has been recorded. As a routine procedure this is useful not only in simplifying the correction of sedimentation rate, but it is a quick and accurate means of discovering whether the patient has anemia or polycythemia.. Furthermore, from the thickness of the layer of leukQcytes and platelets which is found above the layer of packed red corpuscles, one may gauge roughly the quantity of these corpuscles in the blood. Finally, the icterus index may be measured by comparing the color of the plasma in the hematocrit with the color or a series of tubes containing potassium dichromate solution in various dilutions. 9 In infants and young children, when venipuncture is not practical, a modification of the above method may be employed. The heel or finger is punctured deeply enough to secure a free flow of blood.. With a capillary pipet blood is transferred quickly in successive small amounts to a small test tube containing approximately one-tenth the amount of oxalate mixture used for 5 cc. venous blood. Thorough mixing of blood and anticoagulant must be secured. After a little more than 0;5 cc. of blood has been obtained, it is transferred to the hematocrit, which is filled only half way. Sedimentation is then recorded in the usual manner, following which the blood is centrifugalized. To obtain the volume of packed red cells per 100 cc. of blood, the reading must, of course, be multiplied by two. NORMAL SEDIMENTATION RATE.
PHYSIOLOGIC VARIATIONS
The normal sedimentation rate differs according to the method used. It is therefore essential that a standard pro"' cedure be followed and the normal for the method employed be known. In health a slight interindividual variation in sedimenta~ tion rate occurs, and there is also a distinct difference in rate between the sexes. Thus, when the technic described was em-
1544
MAXWELL M. WINTROBE
ployed, the sedimentation rate in healthy men averaged 3.7 mm. at the end of one hour (0 to 6.5 mm. in 86 per cent of the subjects) while in healthy women it averaged 9.6 mm. (0 to 15 mm. in 86 per cent). This difference, however, is largely accounted for by the quantitative difference in corpuscles in the blood of men and women. When these sedimentation rates in 136 men and in 100 women were "corrected" to a volume of packed red cells of 47 cc. per 100 cc. of blood, the sedimentation rate in 72 per cent of the men and women ranged between 0 and 6 mm. at the end of one hour, and in an additional 16 per cent, it was 7 to 10 mm. These values may be considered the limits of normal. As has already been mentioned, sedimentation is accelerated during pregnancy. From about the tenth or twelfth week there is usually a gradual increase in rate which does not return to normal until the third or fourth week postpartum. A slight fluctuation of the rate of sedimentation occurs in relation to the menstrual cycle, but the changes are so small that, from a clinical standpoint, they are of no significance. Other factors, such as the ingestion of food, or. short violent exercise, are of no importance. VARIATIONS IN SEDIMENTATION RATE IN DISEASE
Methods for the determination of sedimentation rates and the variations under physiologic conditions have been discussed in some detail because, unless the technician performing the test and the physician interpreting the results are fully cognizant of these details, important errors may be introduced and the results in consequence become misleading. When the method is understood, however, the test becomes an extremely valuable aid in almost all phases of clinical medicine. In the interpretation of the sedimentation test it is essential to bear in mind that it is a nonspecific reaction which may be compared with the temperature chart, the pulse rate and the leukocyte count, in that it gives information of a general character. It is in fact less specific than the leukocyte count when the differential examination of the white corpuscles is included under the latter head, for then the leukocyte count may offer a clue to the nature of the disease . . ,.Itway be asked what purpose there is in the sedimentation
BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
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test if it affords information of the same type as the temperature ·and pulse record and the leukocyte count. In answer it may be pointed out that in many disorders and even in certain stages of febrile diseases, there may be no fever or tachycardia and the leukocyte and differential counts may be essentially normal; yet in many of these instances the sedimentation rate may be increased. The sedimentation test is, thus, complementary to these valuable clinical tools. Like them it is a measure of the presence and intensity of morbid processes within the body. Sometimes an increased sedimentation rate is found and nothing can be discovered to explain this increase. So little is still understood about the exact nature of the sedimentation reaction that a complete definition of the circumstances under which alterations in sedimentation rate may be expected to occur cannot be given. Yet, if the sedimentation rate in any patient is found repeatedly to be increased, the careful physician will seek to discover the cause. The sedimentation rate is increased in all acute general infections. The degree of increase tends to parallel the severity of the infection. Although an accelerated rate generally does not appear as soon as elevation of temperature, the abnormal rate subsides more slowly and for this reason it may serve as a guide to the condition of the morbid process after fever and tachycardia have disappeared. When complications develop, the sedimentation rate is further increased and this fact may serve to indicate the onset of a complication before its nature and site have been determined. In localized acute inflammatory conditions, variations in sedimentation rate depend on the nature and severity of the morbid process. In simple catarrhal inflammation, such as acute catarrhal appendicitis, simple rhinitis and bronchitis, the sedimentation rate tends to be normal whereas in localized acute suppurations, such as pelvic inflammatory disease, suppurative mastoiditis or sinusitis, there is a pronounced acceleration of sedimentation. In chronic localized infections the rate varies with the extent and nature of the infection, normal values often being found in chronic tonsillitis, whereas increased rates occur in chronic bronchitis, tuberculosis, syphilis, nephritis and liver abscess. Generally speaking, sedimentation rate tends to be normal in
MAXWELL M. WINTROBE
uninfected benign new growths whereas it may be increased when a malignant tumor is present. The latter, however, is not necessarily the case and the factors which probably determine whether the sedimentation rate is accelerated or not include the anatomic character of the growth, its vascularity, the tendency to break down, the degree of resorption of tumor tissue, the degree of reactive inflammation, and the location as well as the presence of metastases. 4 SEDIMENTATION RATES IN VARIOUS DISORDERS
The sedimentation reaction has been demonstrated to be of particular value in following the course of a number of disorders. It will be of interest to discuss these in detail for they illustrate the peculiar advantages of this test. That the sedimentation test is extremely valuable in phthisiology is attested by numerous reports. In pulmonary tuberculosis, variations in sedimentation rate reflect the intensity of the morbid process more accurately than the pulse, temperature, weight, sputum, symptoms. or physical signs. At the Trudeau Sanatorium" it was found that in some instances an increase in rate gave warning of relapses even before new shadows were found in the roentgenogram. No better claim for the test than this can be made, for the value of the roentgenogram as an index of the condition of the tuberculous patient is well established. When one considers the expensiveness of roentgenography and the difficulties sometimes encountered in interpretation, as compared with the simplicity of the sedimentation test, the importance of the latter becomes apparent. It does not, of course, offer any evidence regarding the extensiveness of the tuberculous lesion. The sedimentation reaction indicates the intensity and activity of the inflammatory process. In extensive and yet quiescent fibrotic lesions, sedimentation is less than in limited tuberculous processes with marked tissue disintegration. The sedimentation test is ravored by some investigators even in comparison with the various leukocytic indices. The value of the latter depends in large measure on the ability and interest of the observer. Differential leukocyte counts perfornred in a cursory manner by persons with inadequate train-
BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
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ing are of little value. Here again the simplicity of the sedimentation test gives it an important advantage. In patients who are not under constant observation, the sedimentation test is especially valuable when repeated at regular intervals. Again, it is important in those receiving pneumothorax therapy for here collapse of the lung makes physical signs and roentgenograms of less value than usual. A few observers have reported the finding of normal sedimentation rates in the presence of active tuberculous infectidn. Most workers will agree, however, that such a finding is extremely rare and usually represents a technical error. At the same time it must be pointed out that in severely cachectic stages of tuberculosis, the sedimentation rate has occasionally been found to be normal, or at least slower than earlier in the disease. In the study of rheumatic fever, the sedimentation test is gaining an important place. Its value is found particularly in rheumatic carditis, where it is regularly accelerated. Except when associated with congestive heart failure, a decreasing rate nearly always reflects diminishing activity of the rheumatic process and as an index of this the sedimentation reaction is much more valuable than the temperature, pulse rate or leukocyte count. It has been found that an increasing rate presages clinical exacerbation and many clinicians now permit no activity on the part of the patient until the sedimentation rate has returned to normal. . In acute cardiac infarction and in syphilitic aortitis the sedimentation rate is accelerated, whereas in angina pectoris and hypertensive heart disease the rate is normal. In cases of cardiac infarction the test is a valuable guide in deciding upon the duration of rest because in this condition, as in rheumatic carditis, an accelerated rate may be the only evidence of activity of the morbid process. Rheumatoid, tuberculous and gonorrheal arthritis, and other inflammatory diseases of bones and joints are associated with accelerated sedimentation rates, whereas in hypertrophic or osteo-arthritis, the rate is usually normal. For this reason, the test has been used in aiding the differentiation of rheumatoid from osteo-arthritis. In this respect, however, it has not always been found reliable.
MAXWELL M. WINTROBE
In gynecologic practice, the sedimentation test has achieved a position of great usefulness. The principles involving its use are the same as those already outlined. Uncomplicated benign tumors, such as fibromyomata or ovarian cysts, mild infections or quiescent inflammatory disease, are associated with no increase in sedimentation rate, whereas when there is active inflammatory disease the rate is accelerated. Degeneration of benign tumors, twisting of the pedicle of an ovarian cyst, and often malignant disease, are associated with rapid rates. Since the sedimentation rate is not usually increased in pregnancy until the tenth or twelfth week, the test can be used as an aid in differential diagnosis and as an indication of the presence of complications in cases of abortion and ectopic pregnancy. Retention of uninfected products of conception, hemorrhage or infection causes acceleration of various degrees. In acute and subacute salpingitis the rate is greatly increased whereas in acute appendicitis only slight increases usually occur. Subsidence of pelvic infection is associated with return of sedimentation rate toward normal. Many gynecologists have found this fact a useful guide in indicating a favorable time for elective operations. In aiding the study of gastro-intestinal disorders, the sedimentation test has not as yet attained the general application that it enjoys in the fields already discussed. It has been used by some clinicians, however, to facilitate the differentiation of functional from organic disturbances, benign ulceration from malignant, simple diarrhea from enteritis and colitis. The usefulness of the test in these respects is limited. As has already been pointed out, an increased sedimentation rate is not always associated with malignancy and whether or not the rate is accelerated in inflammatory disorders depends on the severity of the inflammatory process. It is of interest to note that in parenchymatous dis,eases of the liver, sedimentation rate may be actually less than normal. This has been explained as the result of failure to form fibrinogen which, as already mentioned, is closely related to the sedimentation reaction. Undoubtedly one of the most important uses of the sedimentation test is in calling attention to the presence of more or l~s occult diseases. As a routine procedure to be performed
BLOOD SEDIMENTATION TEST IN CLINICAL MEDICINE
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as part of the general examination of a patient, the test is very valuable, for an accelerated rate may not infrequently be the sole evidence of the presence of disease and serves in this way as an indication of the necessity for further study of the patient. When the sedimentation test is carried out in the manner here described and the other steps in the use of the hematocrit performed, one obtains, as the result of a minimal expenditure of time and effort, information regarding not only sedimentation rate but also concerning the presence or not of anemia, leukocytosis and icterus. When the sedimentation test is used as a routine proc~dure in the examination of a patient, it must not be assumed that a normal rate necessarily signifies the absence of disease .. Occasionally it is found, although it is very rare, that the rate is normal in spite of the presence of organic disease. Thus among 444 cases at the Diagnostic Clinic of the Johns Hopkins Hospital in which the corrected sedimentation rate was 10 mm. or less, manifest organic disease was found in 8 instances. In· one case of continued fever of undetermined origin the sedimentation was 0 at the end of one hour. In two cases of chronic nephritis with marked renal insufficiency the rate was o and 1 mm., respectively, while in a patient who had active pulmonary tuberculosiS' as well as hay fever, it was 7 mm. Again, in a patient convalescing from amebiasis who still had some elevation of temperature, the rate was 8 mm. Three of these 8 cases illustrated the importance of recording the uncorrected as well as the corrected sedimentation rate. One patient had acute pyelitis with hydronephrosis, another had carcinoma of the descending colon and the third had cystitis, pyelitis and renal insufficiency. The corrected rates were practically normal (8, 9 and 10 mm., respectively), whereas the uncorrected values were markedly accelerated (37, 30· and 39 mm., respectively). In these instances the accelerated rates were masked by the correction for anemia. A study at the Henry Phipps Institute in Philadelphia 2 stresses the importance of paying due regard to sedimentation rates which are found to be accelerated although the general examination of the patient is negative. In 177 of 328 individuals who, after routine examination, had been considered
I55°
MAXWELL M. WINTROBE
healthy or suffering from only trivial ailments, the sedimentation rate was increased. Reexamination of these patients revealed basal nontuberculous infiltration of the lungs in 96, "latent" apical tuberculosis in 32, positive Wassermann reactions in 35, pelvic inflammatory disease in 5, and possible carcinoma of the lung in 1. Only in 8 cases was there no abnormality found on reexamination. Criticism of the sedimentation test arises from a failure to understand the nature of the reaction and, as with all laboratory procedures, from failure to appreciate the importance of attention to what may appear to be trivial technical details. It must be emphasized that slight alterations from the standard technic may cause great differences in results. As has been pointed out, "correction" for anemia may occasionally be misleading. While correction should be made, the uncorrected rate and the hematocrit reading should be recorded as welI' as the corrected sedimentation rate. Again, it may be repeated that normal sedimentation velocity may occasionally be found in the presence of disease. Finally, it must be stressed that the reaction is a nonspecific one referable to QO particular disease and only indirectly of value in differential diagnosis. SELECTED RECENT REFERENCES AND REVIEWS 1. Coburn, A. F., and Kapp, E. M.: Observations on the Development of the High Blood Sedimentation Rate in Rheumatic Carditis, Jour. Clin. Invest., 15: 715, 1936. 2. Cutler, J. W.: The Practical Application of the Blood Sedimentation Test in General Medicine, Amer. Jour. Med. Sci., 183: 643, 1932. 3. Fahraeus, R.: The Suspension Stability of the Blood, Acta med. Scandinav., 56: 1, 1921. 4. Katz, G., and Leffkowitz, M.: Die Blutkorperchensenkung, Ergeb. d. inn. Med. u. Kinderh~ilk., 33: 266, 1928 (Bibliography). 5. Kelley, W. 0.: The Erythrocyte Sedimentation Rate in Estimating Activity in Pulmonary Tuberculosis, Amer. Rev. Tuberc., 34: 489, 1936. 6. Lucia, S. P., Blumberg, T., Brown, J. W., and Gospe, S. M.: The Relation Between the Suspension Stability of Erythrocytes and Various Constituents of Pathologic Human Blood, Amer. Jour. Med. Sci., 192: 179, 1936. 7. Shookhoff, C., Douglas, A. H., and Rabinowitz, A.: Sedimentation Time in Acute Cardiac Infarction, Ann. Int. Med., 9: 1101, 1936. 8. Smith, C. H.: A Method for Determining the Sedimentation Rate and Red C,ell Volume in Infants and Children with the Use of Capillary Blood. Ariler."Jour. Med. Sci., 192: 73, 1936.
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9. Wintrobe, M. M.: Macroscopic Examination of the Blood, Amer. Jour. Med. Sci., 186: 58, 1933. 10. Wintrobc, M. M.: The Erythrocyte Sedimentation Test, Internat. Clinics, J. B. Lippincott Co., Phila., 46th series, 2: 34 (June), 1936 (Bibliography) . 11. Wintrobe, M. M., and Landsbcrg, J. W.: A Standardized Technique for the Blood Sedimentation Test, Amer. Jour. Med. Sci., 189: 102, 1935. 12. Wood, P.: The Erythrocyte Sedimentation Rate in Diseases of the Heart, Quart. Jour. Med., 5: 1, 1936.