the indices and diameters of the erythrocytes and the best haematological criterion of pernicious...

From the 11. (medical) Department of the Kommunehospital, Copen- hagen, Denmark. (Chief Physician: H. I. Bing, M. D.)

I. Historicit1 Notes and Nor111al Vitlllcs. By

STEFAN J0RGENSEN and ERIK J. WARBURG (Late Senior I’hysicisn) (Senior L’hysiciaii).

In deciding whether a case of anaemia should be classed as Addison’s (1855), Biermer’s‘ (1868) anaemia, a disease, moreover, which was described, although incompletely, as early as 1823 by Andral (cited from Eichhorst) and by Crombe in 1822 (cited from Hunter 1907), as well as by Pierry (1840) and Lebert (1853) .(both cited from Eichhorst), great importance will be attributed to the state of the erythrocytes. It may, therefore, be of some interest to bring into prominence sundry points which, even if some of them are well described in the literature, have not succee- ded in establishing themselves in general medical knowledge.

The commonest criterion of pernicious anaemia which is made use of is the increased colour index Johannes Duncan in 1867 was the first to compare the number of erythrocytes and the amount of haemoglobin in distinguishing a form of anaemia (chlorosis). He found that in this disease the individual blood corpuscles contained less haemoglobin than normal.

It is, therefore, right to regard Duncan as the clinical pioneer in this subject, but his technique was poor, especially when i t is recalled that Welcker’s two epoch-making works were published in 1854 and 1864. I n Welcker’s work of 1854 the principles of

’ Bierincr’s origiiiiil paper does not mist iii Copeiihngen bnt is giraii r c r b a t i m iii Eichhorst’s monograph.

110 BTEYAN JBROENSEN AND ERIK J. WARBURG.

clinical haemoglobin determination and a principle of the blood volume determination were laid down, and he also improved Vierordt’s technique for enumerating the blood corpuscles. It is interesting to observe that this genius even in 1854 attempted to produce a clry scale for haemoglobin determination - a method which Tallquist in 1900 was the first to properly succeed with.

In his work of 1864, which unfortunately from practical con- siderations we cannot refer to in further detail, Welcker occupied himself with the volume of the blood corpuscles. Harting had previously estimated their volume at 76 p 3 . Welcker made plaster models of the blood corpuscles with the aid of the microscope, as he thouhgt he could best allow for the central depression in this way. He weighed the models and then by taking account of the enlargement he could calculate the volume as 72 p 3 (which Iater was too small a value).

In the same work (1864) Welcker showed that the blood corpiiscles i n a case of chlorosis in a young girl, were smaller than normal.

Welcker found that the amount of haemoglobin bore an almost constant relation to the volume of the blood corpuscles in the various classes of mammals, and Worm Miiller (1876) discovered that there mas roughly a proportion between the number of blood corpuscles and the amount of haemoglobin in man.

Our commonly used colonr index is due, however, to Hayem, for like Welcker and Malassez he standardised his haemoglobinometer by counting normal human blood corpuscles and denoted the relation between the colorimetrically determined number of blood corpuscles and that found by direct counting with the letter G (valeur individu?lle dps globules).

Hayeni (1878) found that in mild and moderately severe anaemias G was diminished, while in the most severe (pernicious) anaemias normal or even (cf. Hayem 1889) increased indices were observed. I n Hayem’s earlier papers i t had already been clearly laid domn that the colour index (G) is a function both of the size of the blood corpuscies and of the concentration of the haemoglobin contained in them. While Hayem determined the amount of haemoglobin in the blood and the number of erythrocytes he measured the size of the blood corpuscles, and he could not fall to notice that in the severest anaemias specially many large blood corpuscles were present, but as in these conditions he also found (relatively) abnormally many small blood corpuscles and

PERNICIOUS ANAEMIA. 111.

observed megalocytes in other forms of anaemia he did not think that the size of the blood corpuscles and indices allowed him to dram any distinction between the different forms of anaemia.

It is curious that Eichhorst believed that microcytosis was characteristic of pernicious anaemia while Quinke gave prece- dence to poikilocytosis - both these symptoms being found, as is known, in the most widely different f-rms of anaemi2, and i t was, therefore, a very considerable advance when Laache in Christiania (Oslo) University's programme in 1883, showed definitely that the high coiour index and the numerous large blood corpuscles were the commonest finding in progressive pernicious anaemia.

Laache's iilvestigations - important though they were - have hardly had, however, such an influence as Pan1 Ehrlich's (1881) demonstration of the ciose relatioilship oi megalocytes and megalobhats to pernicious anaemia, but from the literature we have perused, Laache's contention regarding this particular point seems to us t o be just as well proved as Ehrlich's.

It seems to IIS, a t this opportunity, worth pointing out that s. T. Sorensen in his thesis: Investinnlions into the number Gf red and white ' blood corpuscles under ph!jsioloqical and patholcgical co,nditions, Copenhagen 1876, maintained that rnegalocytosis was Characteristic of pernicious anaemia. He writes (in italics): ))On the contrary I find the .considerable size of the blood corpuscles fairly characteristic, for I ?taw not observed suclz i n other morbid conditions, and this forms a contrast to what occurs in chlorosis where, as naentioned, 1 often met with blood corpuscles which were smaller than normal)).

This is the first'time we ,have found megalocytosis referred to as pathognomonic of pernicious anaemia - Sorensen, howcver, retreated somewhat from his standpoint in a paper in 1877 - but his name is now almost always absent in references to perni- ciotis anaemia, which is no doubt due to the fa,ct that this thesis written in Danish has shared the fate of many similar ones - to be read by few and cited by fewer, unlike what may usua,lly be said regarding the relation between the number of readers and those who quote the work.

As mentioned, Hayem correlated the mean diameter of the blood corpiiscles with the colour index; he calculated their mean volume assuming that the erythrocytes had the shape of a cylinder the bottom surface of which mas a circle whose diameter was the

112 STEFAN JBRGENSEN AWD ERIK .J. WARBUKG.

mean diameter of the erythrocyte and whose height was 1.5 p. He then found that

the diameter of the normal blood corpuscle was 7.5 ! r ; volume 66 D > > 3 D small D 7.0 ! i ; D 57 @ D , > , > > 6.5 : i ; 2 49

> D D B a , * 5.0 !(; > 42 / i s

He further made the tacit assumption that the amount of haemoglobin per unit volume could not increase above the normal and concluded from this that the quantity of haemoglobin per blood corpuscle, which is proportional to the colour index, was less than normal when the blood corpuscles were small.

Only a few large investigations exist with determination of the colour index and measurement of the diameter of the blood corpuscles; they were made chiefly by Tallquist, Hayem, Chr. Gram, Laache, Engelsen, Schaumann, Capps, Meulengracht, Prince-Jones and v. Boros.

It took nearly 20 years before the significance of megalocytosis gained full recognition. Grawitz, a t the conclusion of the last century, was not able to recognise in i t a practical criterion, nor could Hunter (1907) see the great importance in diagnosis which this symptom possessed, although in his monograph of 1901 he laid considerable stress on the increased colour index.

In recent years there has been complete agreement that the raised index (as a rule the colour index) and the megalocytosis are two of our most important signs of pernicious anaemia and i t is now hardly possible to find any author with haematological experience who will deny this.

It was a curious thing, however, that while the direct micro- metric method for the determination of megalocyt'osis was the one most used in the seventies and the beginning of the eighties (Sorensen, Hayem, Ehrlich, C. Gram, Engelsen), the indirect method - estimation of the colour index - was almost the only one employed in the last decades of the previous century and the first decades of the present one. Love11 Gulland (1907)) however, emphasised very strongly the value of the direct determination of the size of the erythrocytes.

In recent years interest in micrometry has again been increasing - in the anglo-saxon countries especially since Prince-Jones published his valuable contributions in which there was an excellent statistical and clinical investigation of the size of the erythro-

PERXICIOUS A X A E M I A . 113

cytes. If we might be permitted to criticise Prince-Jones on one point, we would say that want of knowledge of the extensive earlier literature has ca,used him to prefer measurement of the erythrocytes in.a dry preparation rather than in a wet condition.

Prince-Jones and the authors influenced by him, Passey and Carter, Shackle and Hampson, Grosh and Stiefel, regard the increase in the mean diameter of the blood corpuscles as characteristic of megalocytosis. In the present work we claim’to show that that percentage number of blood corpuscles which exceeds a definite limiting value is a much safer and sensitive criterion.

In the Germanic countries the interest in the determination of megalocytosis has also been on the increase, ,especially with regard to the studies of Naegeli and his collaborator Brosamlen, the contributions of Martius and Weinburg, the experimental investigations of Seyderhelm, and Zadek’s studies on the haemoly- tic conditions in pernicious anaemia.

Kiimmerer and Wrack and E. Lovy have described methods, the principle of which is a comparison of normal and pathological blood in the same microscopic field in order to decide whether megalocytosis is present or not. I b Freuchen has introduced a similar method in Denmark.

In table I below we have given as many results of the mean length of the diameter of the erythrocytes as i t has been possible for 11s to procure.

The : oldest investigations have been cited from Haller, Milne Edwards, Harting and Welcker. We have tried to see the originals of the latter publications. The letter ad)) indicates that the measurement’was made on a dry preparation and n ~ ) ) , on a wet’one.

In the case of the oldest papers we cannot in all cases vouch for the fact that human blood corpuscles (except Leeuwenhoek’s) \were used, but in the case of Young and subsequent authors there is no doubt about it.

Leeuwenhoek’s measurements were made in the latter half of the 17th. century. He compared the sizes with grains of sand (an erythrocyte = l / l O O sand grain). According to Harting, Leeuwenhoek estimated his sand grains a t 1/30 inch.

The conversion of inches into millimetres has always been made on the assumption that they mere English inches.

Gulliver’s and Richardson’s figures are in inches. .Unfortunately none of Woodward’s photographic measwe-

ments have been accessible to us (Joseph Janvier Woodward: 8-270.150. dctcr vied. Scnndinou. I’ol. LS I’f.

114 STEFAN JBltGESSEN A K D ERIK J. \ V A R B U R G

Photo-micrographs. Blood corpuscles of man, dog etc. Phila- delphia, 1876. 45 photos. 4". Washington, 1876.

The application of photography to micrometry, with special reference to the micrometry of blood in criminal cases. 13 pp. 8". Philadelphia. I. B. Lippincoth and Co. 1876. Republished from Philadelphia Medical Times 1875-1876. See also Transactions of the American Medical Association 1876).

We have not included H. Schmid's measurements of wet preparations (7.4) in the table, because this author suspended the blood corpuscles in salt solution before the determinations, nor have we given S. T. Sorensen,~ measurements (mean 7.6) as the blood corpuscles mere suspended in Hayem's fluid.

The not inconsiderable differences between the different authors' results have various causes. In the early days Milne Edwards pointed out that such small microscopic bodies as erythrocytes are usually observed to have double contours due to optical conditions. On p. 83 he writes: ))Ces mesures doivent 6tre considerkes comme de simples approximations, e t il ne faut pas attacher beaucoup d'importance aux differences qu'elles accusent lorsque celles-ci sont trBs EgBres, 2 ou 3 centihmes de millimBtre par exemple . . .

Cette comparaison doit Qtre faite avec plus cle reserve encore lorsqu'il s'agit de mesures prises chez differents animaux par deux ou plusieurs observateurs. En effet, pour les mesures micro- mktriques, comme les observations astronomiques, il eviste des differences constantes qui dependent de la mani Bre dont chaque observateur procBde dans les operations qu'il effectue, e t les differences, que les astronomes appellent les erreurs personnelles, varient ici suivant que le micrographe a l'habitude de prendre ses mesures en dehors, sur ou en dedans du contour apparent de I'objet, e t suviant qu'il emploie tel ou tel procede de mensuration.))

'hble 1.

Mean Diameter of the Erythrocytes in ,". Leeuwenhoek' . . . . . . . . . . . . . . 8.3 w. Jurin ' . . . . . . . . . . . . . . . . . 13 Stephan Halvs . . . . . . . . . . . . . 7.7 Schrciber . . . . . . . . . . . . . . . 8.3 Dessgeulieres . . . . . . . . . . . . . . 3.2

' Cited from Harting. a * Albrecht von Haller (German edition of 1762).

PERNICIOUS ANAEMIA:. 115 Poilng (1813) . . . . . . . . . . . . . . . 5.0 d. Everard Home (1818) . . . . . . . . . . . 15.0 d. Prevost and Dnmas (1821) . . . . . . . . 6.7 w. Wagner (1833) . . . . . . . . . . . . . 8.0 Valentin . . . . . . . . . . . . . . . . 7.1 C. Schmidt (1848) ' . . . . . . . . . . . . 7.7 w. Robin (1853)' . . . . . . . . . . . . . . 7.3 Friedberg (1852) ', . . . . . . . . . . . . 7.4 d. Harting (1859) . . . . . . . . . . . . . 7.7 w. Gnlliver (1845 and 1879) . . . . . . . . . 7.9 d. Vierordt . . . . . . . . . . . . . . . 7.7 Welcker (1864) . . . . . . . . . . . 7.;-7.9 d and w. Klein . . . . . . . . . . . . . . . . . 7.8 Formad . . . . . . . . . . . . . . . . 7.9 Berchon and Perier . . . . . . . . . . . 8.3 Richardson (1874) . . . . . . . . . . . . 9.2 w.

- - . . . . . . . . . . . . 8 .0d . ?vIalioin (1875) . . . . . . . . . . . . . . 7.7 d. Malassez (1877) (1) . . . . . . . . . . . . 7.6 d. Hayem (1878) . . . . . . . . . . . . . . 7.5 w. Schmid (1878). . . . . . . . . . . . . . . 7.3 d. Gram (1883) . . . . . . . . . . . . . . . 7.8 w. Laache (1883) . . . . . . . . . . . . . . 8.3 d. Schaumann (1894) . . . . . . . . . . . . 7.9 d. Eibe (1899) . . . . . . . . . . . . . . . 7.7 w. Capps . . . . . . . . . . . . . . . . . 7.65 d. Prince-Joncs (1910) . . . . . . . . . . . 7.4 d.

Becker (1915) . . . . . . . . . . . . . . 7.4 d. Biirker (1922) . . . . . . . . . . . . . . . 7.!i d. Ohno (1922) . . . . . . . . . . . . . . 8.0 d. Passey and Carter (1924) . . . . . . . . . 7.0 d. Ponder and Millar (1924) . . . . . . . . . 8.8 w. Rampsou and Shackle (1924) . . . . . . . 7.4 d. Wiechmsun and Schiirmeyer (1925) . . . . 7.9 d and w. Grosh and Stiefel (1925) . . . . . . . . . 7.4 d. v. Boros (1926) . . . . . . . . . . . . . 7.4 d. Jolly (1925) . . . . . . . . . . . . . . . 7.5 d. Jsrgensen and Warburg . . . . . . . . . 7.7 w.

- - (1922) . . . . . . . . . . . 7.2 d.

Although improvements in the microscope have been great since the latter part of tlie fifties when Milne Edwards wrote these lines, and it mill, therefore, usually be possible to sharply locate the edge of the blood corpuscles, the uncertainty caused by

Cited front Milne Edwards. 3 Welcker (1864).

5 ; Rethe. 5 Engelsen, Vaqnez and Jolly.

l l ( i STEBAN J@NGENSEX AKD ERIK J. \\‘ARBURG.

diffraction is nevertheless so great that Ponder, who has studied the accuracy of the measurements in these determinations most thoroughly in recent years, is certainly right when he states that this uncertainty is at least 0.2 CL a t each measurement.

Th. Young (1813) had already noticed that the blood corpuscles do not shrink visibly as regards their surface when they are dried in a thin layer. This was later confirmed by Home (1818), C. Schmidt (1848), Welcker (1864), Wiechmann and Schiirmeyer (1925) and doubtless many others.

This view, however, has not gone unchallenged. Manasseiri (1872), on comparing 29 dry with a corresponding number of wet preparations, found that the mean diameter of the blood corpuscles was less in a dry than in a wet condition 23 tipes, but sometimes the opposite was the case. He, therefore, recommends measurement in a wet preparation - in which the blood corpuscles are in their natural state. Prince-Jones (1920) found that the diameter of the blood corpuscles in a dry preparation stained by Jenner’s method was 0.7-0.8 p less than in plasma.

If one compares the modern measurements of the human blood corpuscles in a dry preparation - especially Prince-Jones’ and those reported from Guy’s Hospital by Passey and Carter, and Shackle and Hampson with Gram’s and our own - i t is found that size determined by these English investigators is less than that of the Danish.

Still another technical point in measuring the erythrocytes must be alluded to. Manassein measured his moist preparations by laying a thin cover-glass dnectly upon a drop of blood and painting oil around the edge to prevent drying.

Buntzen collected the blood in capillary tubes whose ends he sealed, and obtained, on coagulation, a drop of serum with a few blood corpuscles in it. He put this drop in a counting chamber and measured the diameters. Chr. Gram used the Manasskin- Buntzen technique but he introduced the modification of putting a little water in the circular groove of the counting chamber‘ for the purpose of protecting the blood corpuscles against evaporation, but he introduced thereby a hitherto overlooked error. When a little drop of blood is put quite close to distilled water, the water will condense on the edge of the drop since serum has a little less vaponr pressure than water (the decrease in the vapour tension is 113 %). Under ordinary circumstances this will not be observed, as the rate of diffusion in the air and especially in

I’EIWICIOUS A N A I G M I A . 1 1 7

liquids is relatively small and no mixing takes place, but when the systems are so small as those in a counting cha.mber the diffusion paths are also small, and since the rate of evaporation is very great a dilution of the serum in the margin of thedrop will occur. In 1.904 G. Barger used this phenomenon to determine the osmotic pressure of the blood (which is proportional to the reciprocal of the aqueous vapour tension) by inclosing drops of blood and salt solutions of known osmotic pressure in capillary tubes. In such a system the drop which has the highest osmotic pressure, increases in size, and it is thus possible, by measurement under the microscope, to find the sa.lt solution whose vapour tension is equal to that of the blood.

That these considera.tions possess more than theoretical interest we have shown by the foflowing experiment. A counting chamber is furnished, according to Chr. Gram’s procedure, with a thin suspension of blood in serum on the centrd disc and distilled water in the surrounding groove, which condenses on some of the erythrocytes in the edge of the drop.

The diameters of the blood corpuscles in ocular micrometer units (= 1.4 { I ) are given in the table.

Blood Diameter After After Corpuscles Immediately 20 Minutes 70 Yiuntes

I . . . . 5.1 5.4 5.8 11. . . . 4.8 5.1 5 . 2

I11 . . . . 5.0 5.5 r).7 I V . . . . 5.0 5.2 5.3

r-rV i 9 .9 21.2 22.0

In the second experiment - after having fised a microscopic field in the edge of the drop - we measured all the blood corpuscles which were accessible with the ocular micrometer. The result was as follows (ocular micrometer units):

12.35 p. 111.

3.1

5.3 4.6 4 . i 4.4 5.2 5.1 5.1 3.8

43.3

12.50 p. m.

6.0 5.1 5.0 5. ; 5.2 4.9 5.0 5.1

45.4

3.4 -

1.30 p. m. 6.5 5.1 6 .3 6.0 5.G 6.0 5.4 5.2 4.1

50.2 __

118 STEFAN JBILGENSEN AIUI) EltlK J . WA1U3UI1C.

As the sweIling of the erythrocytes only occurs in the edge of the serum drop and practically not a t all in the centre, the mean diameter will only be altered a little, but disproportionately many large blood corpuscles will occur on measurement of a wet preparation when Chr. Gram’s technique is used.

With respect to, the photographic measurements published by Ponder and Millar by which the author thinks he has avoided certain technical faults, we would remark that they give such a high result (8.8 p ) that we do not feel we can accept it; it im- plies that the blood corpuscles (see later) are much thinner than one would otherwise believe.

It seems that on the whole the differences found by various authors are greater with dry than with wet preparations, which is no doubt due to the fact that measurements in dry preparations demand considerable personal skill both in malung the blood film and in the choice of a suitable place for the measurements. On the other hand Biirker’s, Eisbrich’s, Laache’s, Ohno’s, Schauman’s and Prince-Jones’ excellent investigations show that the same investigator can obtain absolutely reliable values by measurements in dry preparations.

The value which most frequently reappears in measurements of wet preparations is 7.7 ,u, and as we also have found this we believe we can state that the true value of the mean diameter of the blood corpuscles (measurement in serum) is

7.7 p.

It may be remarked that Prince-Jones and after him Wiech- mann and Schiirmeyer in measurements of dry preparations have found that the mean diameter of the erythrocytes varies somewhat in relation to meals and sleep. We have always taken the blood 2-4 hours after the morning meal.

Besides meals and sleep vigorous exertion and the type of re- spiration have an influence on the mean diameter of the blood corpuscles (Prince- Jones). In this connection some almost un- known investigations of Th. Eibe made in 1907 are particularly interesting, for he showed that the blood corpuscles in lunatics shrank during the depression phase and swelled up during the exaltation phase.

We shall not attempt systematically to pass in review the measurement technique which has been employed hitherto; in many of the comprehensive handbooks on microscopy such an

I’ERN IC [OUS AN A EM [ A . 119

account is to be found. Harting, especially, has occupied himself with the question, and contributed some critical remarks towards its elucidation.

Whether the blood corpuscles be drawn with a camera lucida (drawing apparatus) as was first made possible by Sommering’s and Amici’s apparatuses, and then the drawing measured like Milne Edwards. Malassee, Jolly and Prince-Jones did, or whether the far older method with the ocular micrometer (Gascoigne [1717], Benjamin Martin [1740]) be employed, the value obtained will be influenced by the accuracy with which the stage micrometer is divided.

Harting and, later, Welcker complained greatly about the want of agreement among the different micrometers; in recent years we have not seen anything about this in the haematological literature. We have checked our measuring ocular against a stage micrometer by Leitz about 15 years old and also against one by Zeiss about 12 years old, both graduated in 0.01 mm. In the calibration we measured the whole of the ocular micrometer (50 division marks) against 30 different divisions spread over the stage micrometer of Leitz and against 19 on the Zeiss micrometer (every 5th division), and we found one division of the ocular micrometer equal to 1.416 [ I for both the micrometers.

The graduation was extraordinarily good; by far the greatest number of the measurements gave 50 ocular micrometer units = 7.0 stage micrometer units. All the measurements lay between 50 = 7.0 and 50 = 7.2. As the division marks of the stage micrometers occupy almost 115 of a division - a t any rate more than 1/10 - the eye is easily led to estimate 50 ocular units = 7.0 stage units (at the magnification employed), whereas i t would per- haps be more correct to estimate 50 = 7.1. If, therefore, there is any error in the calibration it must be that the units were judged just a little too small.

The ocular micrometer divisions proved nowhere to diverge definitely from the mean value.’

An indirect method for measuring the mean diameter of the blood corpuscles mas described by Pijper in 1919. He used a dry preparation of blood corpuscles as an optical ngratingr, and from a measurement of the diameters of the’ coloured rings which arise he calculated the erythrocytes’ diameters.

‘ In calibrating the ocular micrometer, the stage micrometer was covered with serum and a cover-glass as in ordinary measurements.

120 STEFAN .IBRGENSEN AXD ERIK J. WAHBUKC.

We would like to add a few remarks, firstly, historical, about this method. An apparatus on the same principle as Pijper’s was constructed and described by no less a person than ‘Ch. Young more than 100 years before the former made his, and under the name of eriometer for determination of the thickness of wool hairs i t was to be purchased from Mr. Robinson of Devon- shire St., London. Young’s apparatus was simpler and cheaper than Pijper’s but certainly not inferior, and Young himself used it for determining the mean diameter of the blood corpuscles.

Bergansius has investigated Pijper’s method more thoroughly and has tendered a thereotical and practical criticism of it. From his work i t appears that neither the theory of the formation of the coloured rings nor the practical elaboration of the method has been sufficiently worked out for it to be used in the clinic (which Bergansius himself, however, believes can be done). In his monograph Ponder speaks in great praise of Pijper’s method, but he aaserts that the figures obtained diverge systematically from the true ones.

At no small expense to the laboratory of the Hospital De- partment, we have had an apparatus constructed in accordance with Pijper’s sketch in the 1;ancet (1924)) but the results we have been able to obtain have not sufficient accuracy or certainty (dry preparation) among themselves for us to feel confidence in re- commending Pi j per’s technique .

Our measurements were made with a Zeiss homogeneous immersion 1/12, measuring ocular 3, tube-length 155 mm. + re- volver (sometimes however another arrangement).

Wherever possible we have always sharply located the edge of the erythrocytes, and when this could not be done on account of the occurrence of double contours, we have taken the measurement from the outermost edge.l

The blood corpuscles were suspended in their own serum and covered by a cover-glass 24 x 36 mm. in size. The measurement was made only in the middle of the preparation. The measurements were made after the blood corpuscles had been centrifuged down by rapid spinning for about 5 minutes, and the uppermost again mixed with serum.

Ponder and Rfillar think that a large error may be incurred in The measurements were all made by S. J., the haemoglobiii and volume

determinations hy E. \V. aud the counts by the routine assistant of the Depart- ment, Miss G. Jacohsen. Not until d l the determinations had been made did we compare these.

PERK ICIO US AN.4 EML4 1-21

this way, as the large blood corpuscles should be driven down much more quickly than the small so that the biggest corpuscles would lie a t the bottom of the tube. The investigations of Haldane and Lorrain Smith from Bohr’s laboratory, which Ponder accepts, are not good enough to permit such a conclusion being drawn and moreover they are discredited by Bdnninger’s experiments.

In our measurements we have followed Meulengracht’s procedure and have usually been content with measuring 50 blood corpuscles, while Gram and Engelsen, who used practically the same technique - according to Manassein - measured 100. Prince-Jones measures 200-500 (in dry preparations) and v. Boros measures 200 (in anisocytosis up to 800). We are aware that i t would be desirable to measure more than we have done, but on the other hand v. Boros is not right in contending that‘the number measuied is far too small. The experience of v. Boros with measurements made in native undiluted blood does not allow judgment to be passed on the uncertainty of the measurements made with Manassein’s, Hayem’s, Gram’s, Engelsen’s, Meulengracht’s and our own technique - nor, moreover, on the uncertainty in measurements of dry preparations.

The volume determinations were made in thin tubes with a rapidly revolving haernatocrite (4,000 revolutions per minute, 4 tubes to each determination). The centrifuging was always con- tinued until the column of blood corpuscles was lake-coloured (Koeppe’s criterion). The haematocrite method has many times been criticised, for instance by Alder and by Prince-Jcnes, as i t has been thought that centrifuging might force fluid out of the blood corpuscles thus making their volume smaller. This objection is, however, on the face of i t unlikely, for the volume of the blood corpuscles is determined by osmotic forces which are so great that the force with which they are centrifuged down is negligible compared with them. Finally Reich has simultaneously determined the volume by the haematocrite and refracto- meter methods, and Ege has simultaneously employed the haematocrite and Stewart’s colorimetric method. Suzuku has compared viscosimetric and refractometric methods with the haematocrite. All obtained identical results with the latter by these methods. Alder who has criticised the haematocrite, really finds very nearly the same normal values with his viscosimetric method as the haematocrite gives.

All our investigations were made with defibrinated venous

122 STEFAN JBRGENSEN AND ERIK J. WARBURG

blood. The blood was taken in a thick-walled test tube in which there were some glass bulbs. The tube was corked. It was then cautiously inverted several times until coagulation began, when i t was shaken more vigorously, care being taken that the glass beads beat against the cork and not against the bottom of the tube. After clotting was finished the blood was filtrered through gauze.

No precautions were taken to prevent loss of COz, but from the investigations of E. J. Warburg and from those of D. D. van Slyke, Wu and McLean i t can be seen that a t most the blood corpuscles can only shrink 5 % through loss-of CO,, which corresponds to about 0.1 p in diameter.

The blood counts were made in Biirker Turck’s chamber (ob- taided from Zeiss); 32 large squares were always counted, and the dilutions were made, in accordance with the recommendation of Ellermann and Erlandsen, with separate pipettes. The haemoglobin determinations were made with a standardised Haldane apparatus.

Mr Rasmussen, the pharmacological assistant a t the University laboratory for animal physiology, has very kindly undertaken standardisation on two occasions of a Haldane haemoglobinometer. These standardisations, made a t an interval of a year, only differed by 1 % from one another. We then standardised our routine haemoglobinometer twice, with a year’s interval, against this one and the readings also differed by I yo on the two occasions.’ The carbonic oxide haemoglobin tube was kept in the dark. A 0.4 solution of ammonia was used as the diluting fluid. All the haemoglobin determinations were duplicated.

I n Table I are given a number of values for the volume of the blood corpuscles per 5 million erythrocytes, obtained from the literature. They were all estimated with a uhaematocriter.

Capps centrifuged uncoagulated blood undoubtedly for too short a time, although his centrifuge is said, to have made 10,000 revolutions per minute. Bonninger, Meyer-Bisch, Cshki, Froelich and v. Boros all used a haematocrite designed by Bonninger. They were not aware that i t is necessary to centrifuge until the blood corpuscle column is lake-coloured (Koeppe’s criterion); presumably therefore they obtained a little higher result than we do.

Haden centrifuged in a conical tube. He had first diluted the

A standardisation after the termination of these measurements gave. 3 X higher values than the mean of the two mentioned.

PERNICIOUS AXAEMIA. 123

blood with 115 volume l . G % potassium oxalate (said to be isotonic). He also did not ensure that the smallest volume had been attained.

They certainly obtained the smallest volume, for’ their haematocrite revolved very rapidly and for a long time. The apparatus was, however, rather smaller (shorter tube) than that Gram and Nor- gaard and the one we employed.

Gram and Norgaard’s figures relate to blood whose coagulation was prevented by the addition of hirudin. They used the same model of haematocrite as we did and centrifuged until a lake colour was reached. The haematocrite tubes were 10 cm. long.

We regard Gram and Norgaard’s figures as the safest, and their mean value (43 volumes per 5 million erythrocytes) agrees with ours.

de Jong diluted the blood with 3 % isotonic sodium citrate solution and centrifuged 5 minutes a t 12,000 revolutions per minute. He did not obtain the smallest volume, but according to his experiments there was a deficit of 5 %. The method is not to be recommended as not all blood can be centrifuged down in the same time. If we correct de Jong’s figure (45 volumes %) - which in his &sum6 he states to be the mean value per 5 million erythrocytes - we get 43 volumes %.

Kiihnel diluted the blood with 1/10 volume 3 yo sodium citrate solution. He centrifuged to constant volume in graduated tubes containing 5 c. c.

Table 11. Blood Corpuscle Volume in % of the Blood per 5 Illillion Erythro-

cytes (Haematocrite Method). Capps (1903). . . . . . . . . . . . . . . 50.0 Bie arid Maller i1913) . . . . . . . . . . . 41.3 Biinninger (1!1191 . . . . . . . . . . . . . 45.9 . . Meyer-Bish (1919) . . . . . . . . . . . . . 45.0

Froelich (1922) . . . . . . . . . . . . . . . 45.5 Hnden (19’22 and 1923) . . . . . . . . . . 46.0 Gram and Norgaard (1922) . . . . . . . . . 43.0 de Jong (1924) . . . . . . . . . . . . . . 42. i Kiihnel (1925) . . . . . . . . . . . . . . . 43.8 v. Boros (1926) . . . . . . . . . . . . . . . 45.5 Jsrgensen and Warburg . . . . . . . . . . . . 43.0 Osgood (1926) * . . . . . . . . . . . . . . . 42.2

Osgood’s publication did not appear until the present paper had been written, but we have iuclnded the results on account of their ,great accuracy and comprehensive nature (Edwin E. Osgood, Hemoglobin color index, saturation index and volume index standards, redeterminations based on the findings in one

Bie and Moller worked with defibrinated blood.

Csiki (1922) . . . . . . . . . . . . . . . . 45.9

124 STICFAii JMKCENSEN A N D ERIK J. WARBURG.

The erythrocytes’ colour index (index colorimetricus) is estimated from the haemoglobin determination and count of the erythrocytes according to the following formula:

Haemoglobin in % of the ))normal)) Erythrocytes in % of the ))normal))

_ _ - - I . col.

100 % haemoglobin is assumed to be 13.7 gni. in 100 cc,. (Hal-

The formula can be reduced to % Haldane

Er.. 20

dane), and 100 % erythrocytes, 5 million per cmm.

= I. col.

It is generally recognised (cf. H. C. Gram) that normal persons on an average have a colour index 5 1 when a properly standardised haemoglobinometer is used.

As the colour index, in virtue of its derivation, expresses whether the individual blood corpuscles contair!, on an average, the ordinary amount of haemoglobin or not, we must see more closely what determines the haernoglobin content of the blood corpuscles.

As stated, Welcker and Hayem recognised that there was an approximate proportion existing between the third power of the erythrocytes diameter and its haemoglobin content.. This was subject to the proviso that the concentrat.ion of haemoglobin in unit volume of the blood corpuscles was constant; if this assump- fion is valid we can conclude that there is megalocytosis from an increased index and microcytosis from a diminished index.

In the works of Alder, CappP, Cshki, Bonninger, Haden, de Jong, Gram and Norgaard, Meyer-Bisch and the present study i t is, however, shown that the hlood corpuscles are never supersaturated with haemoglobin but are often more deficient in i t per unit volume than normal (de Jong, however, found. constant saturation per unit volume). d n increased (reliably determined) colour index will, therefore, always indicate megalocytosis, while a low or normal index signifies nothing definite concerning the size of the hundred and thirty-seven healthy young men. Arch. Iiit. Xed., 37, 68.‘r-706 (May 1926).

He uses venous blood whose cnagolntion was hindered by the addition of 0.2 % iieutral potassium oxalate; this makes the blood. corpuscles shrink 3.5 % according to Osgood’s statement. He finds tha t the degree of shrinking is independent of the amoutit of blood corpuscles which, however, can only be approximately true, as appears from numerous papers on the oaniotic conditions of the .blood corpuscles (cf. for example, Rich. Ege’s Studier over Glucoseu’s Forhold i Blod and E. J. Warburg’s investigations in 1922). To Osgood’s value of 40.8 volumes % per 5 million erythrocytes we have added 3.5 %.

PERNICIOUS ANAEMIA. 125

blood corpuscles and consequeiztly cannot be the determining jactor in the diagnosis 01 the kind of anaemia.

Recognising this Capps introduced in 1903 the conception volume index:

Volume in % of the ))Normal)) Erythrocytes in % of the ))Normal,,

and believed he could show that this relation afforded a far better indication of whether the erythrocytes were normal in size or not.

Capps’ investigations were subsequently confirmed by Alder, Bonninger, Csaki, Prolich, Haden, de Jong, Meyer-Bisch, Naegeli and Reich in so far as they have shown that the variation in this index is somewhat more characteristic of the different forms of anaemia than the ordinary colour index, but we cannot see that these authors have produced anything fundamentally new.‘

Gram and Norgaard’s and our normal value for the volume of the erythrocytes was:

I Cousiderable interest attaches to tlie fact that the saturation index has nuver been shown to be greater t,hau 1 (apart from analyt,ir:il uncertainty).

lliirker has claimed to show that there is a proportionality betweeii the surface of the blood corpuscles and the amount of haemoglobin present in them. Quite apart from the fact that Biirker did not p:ty sufficient attention to the diameter detcrmiiiatious carried out by unmerous other authors (Carl,* Formad,* Gulliver, Kleinebergcr,* Lange,* Manasseiu, Schanz * and Welcker), b u t practically only relied ou his eollaboratora’, Eisbeich’s aud Ohno’s measurements, the latter, as pointed out by Ponder, do not constitute defiuite proof of’ Biwker’s assumptiou.

Biirker’s calculiitiou of the surface of the blood corpuscles rests 011 the nssumptiou . that the snrface cotist,ituted by the sides of the blood corpuscles call lie ueglected, but this is cerhiuly not correct. Thus Knoll has shuwn tha t with human blood ,CoriJllSCieS ail error of at least 27 % is iucurred in this way, aud Pouder :iud subsequently Neohansen have iutroduced corrections :or the concavity of t,he surface of the blood corpuscles.

The, most importaut error, however, arises from the fact that the shape of the blood corpuscles in differeut animals is uot snfficiclltly well kuown, aud i t is certaiuly riot right to assume tha t the total surface stands i n the sanie rela- tiou to the two largest surftices of the erythrocytes in all cases (cf. de Jong).

The mere fact that i n inicrocytosis (in nian) where the blood corpuscles have a ielntively large surface we do uot find an increased saturatiou index and in megalocytosis we do uot co,)astniatly find a low one, argues strongly against 15urker’s theory.

Moreover the physical sigriific:iuce of the fact that the haemoglobin is assumed to be united with t,he surface of the lilood corpuscles is retidered difficult owiug to) 32 % h:iemoglobiu beiug present in them, aud lietweeu ‘ i s and ‘/i of‘ their volnme must, therefore, be full of haemoglobiu. Siuce Adair has shown that haemoglobiu is solable enough iu a watery solotiou of sal t for i t to Occur iu solution iu the blood corpuscles, there is hardly auy reason to uphold Biirker’s theory whose consequences are a t variance with ou1 knowledge of the osmotic activity of the haemoglobiu in the blood corpuscles (cf. E. J. Warburg; viln SlSke, Mu and hlclean).

Cit,ed from Eisbeich. _--____

126 STEFAN JBltGENSEN AND IFRIK J. \VAR13UKC:.

5,000,000 erythrocytes corresponds to 43 volumes ”/o erythrocytes. From this we obtain:

Volume % Erythrocytes - Millions of Erythrocytes

Volume Index = - 0.116.

One is prompted by Capps’ work to introduce still another index - the saturation index (Haden). Such a value has also been evolved in a rather different manner by Alder, Bonninger, P. Drucker, H. C. Gram and Norgaard and by Naegeli. We have used the following formula:

% Haldane Volume % Erythrocytes

Saturation Index = _ _ _ _ - ~ _ _ - - 0.43

which follows naturally from the rule referred to. A low colour index may occur in the following cases:

(1) Saturation index = 1, therefore, volume index is less

(2) Saturation index less than 1; the volume index may then than 1 (and there is microcytosis).

be normal, increased or decreased.

What now is the connection between the size of the erythrocytes and the volume index? When we began our work this question had never been thoroughly investigated; in the meantime an article by v. Boros has appeared in the Wiener Arch. f. inn. Med., Feb. loth., 1926, and he takes up the question on parallel lines to ours, in that he only deals with the conditions in normal persons and in chronic congential jaundice occurring in families. We shall shortly revert to this paper.

I n his monograph, ))The Erythrocytes and the Action of simple Haemolysinso, published in 1924, Ponder states that the blood corpuscles are shaped like biconcave discs whose greatest thickness is 30 % of the greatest diameter and whose least thickness is 314 of the greatest. The concavity occupies about half of the cell’s diameter. On the basis of these suppositions Ponder now calculates the cell’s volume and arrives a t the following simplified formula:

Volume = - m A3 2 5

where A is the radius of the erythrocytes. The mean diameter of the blood corpuscles can be calculated

from this formula. If we substitute the accurately determined

I’ERXICIOUR ANAEMIA. 127

value 86 p3 for the volume of the blood corpuscles] we get a mean diameter of 8.2p. This value, as is seen from Table I, is, however, improbably large and on the whole i t is very doubtful whether Ponder’s impressions of the shape of the erythrocytes are sufficiently well founded to support such a calculation (see footnote on p. 125).

Bul; we can go another way to work and merely assume that the blood corpuscles are mutually similarly formed and we can then calculate a mean diameter from the volume by the following formula:

Calculated mean diameter = normal mean diameter x $7. volume.

If we now compare the calculated diameter with the mean diameter found we obtain, on the assumptions made, the following information :

shape.

than normal.

Calculated = found mean diameter:

Calculated greater than found: blood

blood corpuscles of normal

corpuscles relatively thicker

Calculated less than found: blood corpuscles relatively thinner than normal.

In the article mentioned v. Boros has carried out an investigation with a similar object, but in the development of his formula he has made certain assumptions and corrections, but the point he seems to overlook is that they only serve to hide the fact that like ourselves he is working with an empirically determined volume and mean diameter, and, without adequate knowledge of the shape of the blood corpuscle, is only committing himself to the postulate about uniform structure.

I n this calculation an approximation has been made which does not seem to have been clear to earlier investigators. It is assumed that the cube root of the mean erythrocyte’s volume (similar shape being granted) always bears the same relation to the mean diameter of the largest surface of the erythrocytes.

If all the erythrocytes in the same person were of the same size the relation would hold, for then we should have:

This fignre is based on 5 million erythrocytes occnpying 43 X of 1 cmm.

128 FI'XFAX JBRGENBEN AND ERIK J. \VARBURG.

where k is a constant depending on the shape of the erythrocytes and the unit of measurement [cf. Welcker (1864), Hayem (1878), Biirker (1922, 1925), Ponder (1923, 1924), and for the analogous case with the surface, Welcker, Biirker, Knoll and Ponder], while n is the measured number of erythrocytes, V is the volume found (corresponding to n erythrocytes) and D, the diameter required.

When the erythrocytes are of different diameters we obtain, for the sum of their volumes

k3 ((D, + aJ' + (D, + aJ' + . . . (D, + an):'),

where the condition is that a1 + a2 + . . . a, = 0.

Developing the formula further we obtain:

k 3 ( n D 1 3 + 3 ( a I + a , + . . . a , ) D , " C ( a , 2 + a ? 2 + . . . a , , 2 ) D , + + (a13 + + . . . an3)).

The second term of the suni in brackets is equal to 0 and the fourth term can with close approximation (for a large number of measurements without approximation) be put equal to 0, but. the first and third terms are positive.

a12 + + . . . a,, = [a2]

is the sum of the squares of the deviation from the mean diameter. Provided that the distribution follows the binomial error lam, we get, when the variability coefficient is denoted by V, in accordance with the definition of this value:

or with sufficient accuracy

and the average volume then becomes:

n

PERNICIOUS ANAEMIA. 129

I If now we calculate $lie .average hameter from the equation,

we obtain too large a diameter and the error is then 3 .

D l ( f i + - 1). In table 111 we have estimated how much greater the calcu-

lated mean diameter is than the true one for differentsizes of the mean.diameter:and variability constant.

Tnble II1.

Correction to be' Subtracted f r o m the Meun Calculated Diameter. K e a n D i a m e t e r

"'ri'bility 'Oefficient 5.5 6.0 6.5 7.0 7 5 7.7 8.0 8.5 9.0 9.5 10.0 6 . . . . . . . . . . 0 0 0 ' 0 0 0 0 0 0 0 0

10. . . . . . . . . . 0.1 0.1 0.1 0.1 0:1 0.1 0..1 0.1 .0.1 0.1 0.1 15 . . . . . . . . . . 0:1 0.1 0.1 0.2 0.2 0.2. .0.2 0.2 0.2 0 .2 0.2 20. . . . . . . . . . 0.2 0.2 0.3 0.3 0.3 0.3 0.3 0.8 0.4 0.4 . 0.4 25 . . . . . . . . . . . . . 0.3 0.4 0.4 . 0.4 0.5 0.5 0.5 0.5 0.6 0.G - 0 . 6

To 'be absolutely accurate the correction ought to be calculated with reference to the calculated mean diameter and not in relation to the found one. We have omitted making this simple converdion of the corrections because neither the direct diameter measurements nor the .volume index determinations are accurate enough to make this final correction necessary. Moreover the fact that the shape. of the size-distribution curve of the erythrocytes i s not strictly binomial - and particularly under pathological Condi- tions - renders the 'correction rather uncertain. We have practically avoided the small inaccuracy' just mentioned by. making the corrections relative to the directly (by micrometer) determined diameters.

In table I T T we have recorded 7 persons whom we regarded.as normal from a haematological standpoint. Besides the directly determined values the calculated indices are entered in this table as well a's the estimated value of the average blood corpuscle's diameter. For this calculation .we used the formula

:I-______ Mean Diameter = 7.7 'p 1. Volume.

9-270458. A c t a med. Scnndiiraa. I'd. L X V I .

130 STEFAN JBRGENSEN AND ERIK J. WARBURG.

The calculation can be made simply with a slide rule or with a table of cube roots or logarithms, but to further simplify the calculation and the determination of indices one of us in a later contribution has published a nomogram for calculating these values.

The table also contains the mean value calculated in the ordinary way and lastly the variability coefficient of the erythrocytes, this being 100 times the standard deviation divided by the mean diameter.

The calculation of the mean error of the mean value and variability coefficient is based on the assumption that the diameters of the erythrocytes are distributed according to the exponential law of error which agrees fairly well with data (P. Heiberg, Prince- Jones, 1922) obtained under normal conditions. I n anaemias - especidly in the megalocytic - the distribution curve is asymme- trical (Prince-Jones, 1922) and the figures given, therefore, only afford a rough approximation for determining the variability (anisocytosis) and uncertainty of the mean figure.

I n the last column the percentage of the blood corpuscles whose diameters were equal to or greater than 8.6 p. is given.

The mean value of the colour index was 0.94.l This is presumably due to the fact that the standardisation was done against a haemoglobin determination made with a ferricyanide method for estimating the combined 0,. D. U. van Slyke and Stadie have shown that this leads to figures which are about 6 % too low. Gram and Norgaard’s haemoglobinometer was standardised by exhausting with a mercury vapour pump and direct analysis in Petterson’s apparatus. (The determination was made by E. W.). The volumetric index varied between 0.93 and 1.06 with a mean value of 1.00. The saturation index coincided with the colonr index, 0.94; the fact that the former was not 1.00 is due to the same conditions which caused the deviation of the colour index from unity.

In considering our results i t must, therefore, be remembered that our haemoglobinometer has presumably given a reading which was 6 % too low and that the normal value of the colour index and the saturation index of this work is 0.94. The mean diameter is 7.7 varying from 7.5 to 8.0, which lies within the bounds of

IJsiug our later haemometer correction the iiidex becomes 0.976. We have raised the meu i value 7.65 to 7.7 in view of the possibility,

inentioiled on 1). 119, tha t oiie of our scale divisioiia being = 1.416 :L is little too low.

PERNICIOUS ANAEMIA. 131 Table IT.

Normal Persons. - -

z ?

- 1 2 3 4 5 6 7

r m E P B

W 3 P

h

(D v - 98

105 105.: 94

98 94

109,

I /

43.8 45.6 1.0’ 48.3 42.5 0.9 49.1 41.3 0.8 42.5 40.2 0.8 48.0 43.71 0.9 42.8 45.5 0.9 44.3 42.61 0.9

-.

2 ? F ct 3.

- 1.0( 0.9: 0.91

0.91 1.0:

1.01 0.9!

- - B 6 e, % a

5

2

U

ti 5’

cc m - 7.8 7.7 7.6 7.5 7.7 7.8 7.7

1 Measnrements

7.5 7.4 I 8.r 7.; 7.4 0.05 8.1 7.8 7.5 0.08 8.( 7.7 7.5 10.06 8.1

~

Mean 7.65 = (7.7)

-

s ! - 5.g 6.’ 6.1 6. 5. 6. 7.

the figures given in the literature. The variation in the figures is rather wide and is partly due to the real error, as the uncertainty of the mean value is about 0.2 p to judge from the estimation of the error when only 50 blood corpuscles are measured.

The calculated mean diameter varied between 7.5 and 7.8.

Corrected Calculated Mean Diameter in !L

7.6 7.8 7.1;

7.5

7.8

- - I . ,

- _ (,,

Measnred Nean Diameter in !L

8.0 7.9 7.8 7.5 7.5 7.4 7.4

Difference in p 0.4 0.1 0.2 0.3 0.2 0.1 0.3

Mean 0.23

The maximum deviation was 0 .4 ! I and the minimum 0.1 p. A deviation of more than 0.3 $1 is necessary between the calculated and the measured diameter before concluding that the average erythrocyte has an abnormal shape.

In diseases where the variability coefficient for erythrocytes is

132 STEFAN JBRQENSEN AND ERIK J. WARBURG.

large the deviation should even be in the neighbourhood of ’la !I to allow of a definite conclusion. In the 5 cases where the variability coefficient was calculated it was 8 % or less. W e never found over 8 % large blood corpuscles, the criterion for which was that they should have a diameter 2 8.6 [ I . It will later be shown that we lay great stress on the determination of the degree of the megalocytosis and we will, therefore, take some investigations from the literature for comparison with our figures.

Hayem divided the blood corpuscles into 3 groups af which the one with the largest corresponded very closely with our group of large blood corpuscles. He found 75 % normocytes, 12 % megalocytes and 12 % microcytes.

Much the biggest and most thorough investigations in this field are contained in Chr. Gram’s thesis. Gram reckoned those which had a diameter greater than or equal t o 8.65 CL as large blood copuscles, but as his mean diameter was 7.8 p, thus 0.1 ,LI

greater than what we found, his megalocytes of 8.65 ,LI and over should undoubtedly be comparable with our 8.6 p and over. ,

On investigating 20 normal persons Gram found an avefage of 13 % megalocytes with a minimum of 4 % and a maximum of 23 %. The last figure was only reached once, the next highest figure being21 %.

As will be remembered, however, Grams measurements suffer from a technical error which especially makes the number of megalocytes too large.

The following figures have been calculated from Prince-Jones’ outstanding work of 1922.

The mean diameter (dry preparations 500 cells in each) was 7.2 in 20 normal persons. He divided the corpuscles into groups with increments of 0.25 p. If we count the number of blood corpuscles in the group 8.25 ;L and over - which will very closely correspond to our megalocytes - we obtain,

Megalocytes as a percentage of the erythrocytes

. . . . . . . . . . . . . . . . Mean . 311~ :& Maximum . . . . . . . . . . . . . . . 8 % Minimum . . . . . . . . . . . . . . . 2 76

which i t will be seen are much lower figures that quite correspond with ours.

In measurements in wet and dry preparations in 14 persons,

PERNICIOUS ANAEMIA. 1.33

Ohno and Gisevius‘ found a mean of 8.0 p (women 8.0% men 7.88). I n the group 8.75 and over, in precentages of the erythrocytes, the

Mean . . . . . . . . . . . . . . . . . 6 % Maximum . . . . . . . . . . . . . . . 13 % Minimum . . . . . . . . . . . . . . . 2 %

If the group 8.75 to 8.99 be reckoned, only with half the value

Mean . . . . . . . . . . . . . . . . . 5 % Maximum 9 % Minimum . . . . . . . . . . . . . . . . 1 yo

we get

. . . . . . . . . . . . . . .

In measurements in wet preparations (200 cells in each) in 22 normal persons v. Boros found a mean of 7.5 [ I . Erythrocytes whicB were equal to or greater than 8 ci gave as

Mean . . . . . . . . . . . . . . . . . 14 % Maximum . . . . . . . . . . . . . . . 20 % Minimum 7 % . . . . . . . . . . . . . . .

. It can be inferred from this that the number of erythrocytes which are above 8.4 ,LL (7.5 + 0.9) are considerably less.

Laache’s, Schauman’s and Becker’s measurements are not suitable for a comparative study on account of the roughness of their grouping.

From this part of our investigation we believe we can assert that normally there are not 15 % erythrocytes whose diameter

Since this was written a paper by Wiechmann and Schiirmeyer has appeared: Schwankungsbreite und Schwanknngsart der Durchmesser menschlicher Erythro- cyten (Dentsch. Arch. f. klin. Med., 151, 257-265, June, 1926). It takes the form of a criticism of Obno and Gisevins’ paper in as much as W. and S. claim to show tha t the difference between the largest and smallest erythrocytes in normal persons is only 1.1 {L while 0. and G. give 3.2 p. Without doubt this figure is by far the best as i t agrees with numerous statements in the literatnre; only Malinin and Schmid have as small differences as W. and S. We consider tha t the want of agreement between W. and S. and 0. and G. illustrates excellently the danger of measnring the corpuscles i s a dry preparation, and the authors undoubtedly unwittingly make a selection when they say: DDSSS nnsere lfessongen ohne irgendeine Grsssenwahl und nur an einzeln liegenden und kreis- runden Erytrocyten vorgenommen wnrdon, ist selbstverstandlich.~ The mean diameter by W. and S. is, as shown in Table I, 7.9 p (20 persons). They found no erythrocytes with a diameter above 8.i4 u .

They refer to the measnrement curves of two cases of pernicious anaemia where the mean diameter was unchanged bnt where there was an accumulation of megalocgtes.

134 STEFAN JBRQENSEN AND ERIK J. WARBURG.

is 0.9 ci greater than the mean diameter, or that normally - according to the best measuring method - there are less than 15 'X, erythrocytes which have a diameter equal to or greater than. 8.6 ,u.

Summary.

1.

2.

3.

4.

A historical account of our knowledge of the indices and diameters of the red blood corpuscles is given.

X. T . Siirensen seems to be the first (in 1876) to have recognised that meyalocytosis i s characteristic of pernicious anaemia.

The relations between colour index, volume index, saturation index and diameter of the blood corpuscles are developed.

It is shown that from a determination of the volume index and of the mean diameter of the blood corpuscles we can infer their thickness.

5. Normal individuals have less than 15 yo erythrocytes which have a diameter equal to or greater than 8.6 /(.

The Literature is subjoined next paper.

the indices and diameters of the erythrocytes and the best haematological criterion of pernicious...

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