Actn l e d i r a Scsndinavicn. Vol. CXLVI, f‘asc, 111, 1963.

From the Central Laboratory, Sahlgren’s Hospital, Gothenburg, Sweden.

Studies on Fnctom Influencing Erythrocyte Sedirrienttrtioii Rate.

BY E. H. MARTENSSON and H. A. HANSEN.

(Submitted for publication January 8, 1963.)

Introduction. The present paper deals with comparisons between erythrocyte sedimentation

rates (SR) in undiluted blood and in different citrate dilutions of blood, and with the r61e played by the red corpuscles as well as by the plasma and electrolytes. Furthermore, the influences of some physical factors (tube width, tube length, aggregation of red cells and cell packing) are discussed, and some clinical aspects concerning the SR in polycythemia and in anemia are touched upon.

One phenomenon, however, has been received special attention, namely the so- called weversed SRb. In a previous, unpublished investigation using Heller and Paul’s (1934) oxalate mixture as an anticoagulant we made a comparison between sedimentation rates in: 1) oxalated blood diluted with sodium citrate solution in the proportion 4 : 1 (SRocB), 2) blood diluted with citrate solution according to Westergren (1924) in the proportion 4 : 1 (SRCB), and 3) oxalated whole blood (SR,,,). Between the two first-mentioned procedures there was no statistically significant difference, whatever the magnitude of the SR. The third procedure gave SROwB values which were higher, in cases of normal and moderately in- creased SR, than the values obtained with diluted blood. For high SR values, however, the opposite was the case, i. e. SR,,, was lower than SR of diluted blood. This phenomenon will be called weversed SRn.

Reversed SR has previously been observed by several authors, as for example Pinner, Knowlton, and Kelly (1928), Westergren (1937), Strom (1938), Kaulla (1939), Nielsen (1942), ustner (1942), and HorBnyi (1943). All the investigators mentioned, although using different anticoagulants such as potassium oxalate, sodium citrate, or heparin added to whole blood, or, as HorBnyi did, using cel- loidin covered tubes, have on the whole obtained the same results. ostner and HorBnyi have closely studied the causes of reversed SR, and their interpretations will be discussed later. As some remarks and objections can be made especially to HorBnyi’s conclusions, we felt it important to re-investigate the problem.

STUDIES ON FACTORS INFLUENCING ERYTHROCYTE SEDIMERTATION RATE. 165

Experimental. 1. Effect of Varying Amounte of Citrate Solution Added to Whole Blood.

3 Dilution Curves B.

A comparatively large amount of blood (40-80 ml) was collected through veni- puncture into flasks containing Heller and Paul’s (1. c.) dried oxalate mixture (4 mg solid potassium oxalate and 6 mg solid ammonium oxalate for every 5 ml of blood). This mixture has the advantage of not altering the red cell volume. The oxalated blood was then mixed with iso-osmotic‘ citrate solution in the pro- portions 9 : 1, 8 : 2, 7 : 3, etc. down to 2 : 8. The sedimentation rate was deter- mined a t room temperature with undiluted blood, as well as with the different blood citrate mixtures, using Westergren’s technic with readings after one hour. The results are presented in Fig. 1, where the SR values (averages of a t least two, usually of more determinations) are plotted against the ordinate, and the per- centage of whole blood in the mixtures against the abscissa. The different SR values for each blood sample are linked to form a ))dilution curve)). The dilution used in the Westergren method, i. e . 80 per cent blood and 20 per cent citrate solution, is specially indicated with a vertical line.

As can be seen from Fig. 1 the curves are of two main types. The one group (Type I) comprising normally and moderately settling bloods is characterized by a steady drop in SR values with gradually increasing dilution. I n the other group (Type 11), representing rapidly settling bloods, the dilution curves first rise more or less steeply, reach a maximum, and then quickly fall off t o low values. This shape of the dilution curve illustrates the phenomenon of oreversed SRn. The ver- tex has never been found beyond 60 per cent of whole blood. Westergren (1924) has previously published dilution curves similar to the first-mentioned group only, but none with SR,, exceeding 60 mm in one hour was presented.

Ostner (1. c.) published dilution curves (although limited to the range between 100 and 80 per cent of blood in the blood citrate mixture) also for rapidly settling bloods, and encountered reversed SR even with SRcB values of only 11-20 mm in one hour, in this respect diverging from our findings as illustrated in Fig. 1.

To confirm our results we performed 106 comparisons between SR of oxalated whole blood and SR carried out with the Westergren dilution technic (SR,,,) as a reference method. Increasing frequency of reversed SR was recorded a t SRocB values above 65 mm, and a t values above 100 mm reversed SR was seen in 83 percent of the cases. A comparison between (jlstner’s material (100 cases) and ours is presented in Fig. 2, where the occurrence of reversed SR is computed in per cent of the total number of estimations within the ranges examined.

The differences are probably not due to divergencies between the two materials, as btner’s material originates from internal, epidemic and phthisic patients and ours from internal and to some extent from surgical patients. A more acceptable

3.0 per cent sodium citrate (Na,C,H,O,. 2 8 , O ) solution, isoosmotic with blood (Gram 1928, Hirschlaff 1936, Lund, Nielsen, and Pedcrsen-Bjergaard 1947) has been used throughout this investiga- tion instead of 3.8 per cent according to Westergren (1924).

E. H. MAXTENSSON AND H.

I A .

- 10

% whole blood in blood-citrate mixture

Fig. 1. Fig. 2. Fig. 1. Variation in SR on dilution of oxalated whole bloods of different settling rates with varying amounts of citrate solution (udilution curvesu). The vertical line represents the degree of dilution with Weatergren’s technic. - Fig. 2. Frequency of weversed SRP in 6stner’s material (-----) and in the

present authors’ material ( - - - . - - -), referred to the SR of citrated blood.

explanation could possibly be found in a difference in the red cell volume, which markedly influences the occurrence of reversed SR. This, however, as estimated from the hemoglobin values, is about the same in the two materials. The influence of erythrocyte volume will be discussed in detail later.

The discrepancy is rather t o be found in dissimilar technics. Thus usher uses heparin to prevent coagulation. This substance may change the stability of protein solutions (Albertsen and Heinteelmann 1950), and will a t higher concentrations form complexes with some globulin components and fibrinogen (Hoch and Chanu- tin 1952). Concerning SR heparin has also been considered to affect the red cell aggregation and give rise to capricious sedimentation - at least at higher con- centrations (Strom 1. c., Kaulla 1. c., Nielsen 1. c.).

From Horhnyi’s cases of reversed SR (23 tests) an average SRGB value of 115 mm in one hour can be interpolated, indicating occurrence of reversed SR only for rapidly settling bloods. This agrees with our findings showing a corresponding mean of 107 mm in one hour,

Horhnyi, however, ascribes the reversed SR to a special citrate effect. TO ex-

STUDlES OX FACTORS INF1,UESCINQ ERYTIIROCTTE FEDIBIENTATIOX RATE. 167

d u d e the possibility that oxalate has properties not shared by the anticoagulants mentioned, dilution experiments have been performed on citrated as well as on heparinized whole blood. Similarly shaped dilution curves were obtained, in- dicating no essential difference between the anticoagulants in question.

2. Effeot of Eleotrolytee.

It is well known that electrolytes exert a suppressing effect on the aggregation .of red corpuscles (Nasse 1836, FBhreus 1921, Oliver and Barnard 1924, Rourke and Plass 1929, Swedin 1936). Most of these investigations were carried out under conditions not directly comparable to those existing in the determination of SR in the clinical laboratory.

To ascertain the influence of a slight increase in electrolyte content caused by addition of an anticoagulant to whole blood, a number of experiments were made. In a large centrifuge tube covered with a thin film of celloidin (HorOnyi 1. c.) 40-50 ml of blood was collected and immediately divided into four parts. To three of them Heller and Paul’s (1. c.) mixture of solid potassium and am- monium oxalate (2 mg per ml), solid sodium citrate (4 mg per ml), and heparin (0.1 mg per ml) were added respectively, the fourth was left in its original state without any addition of anticoagulant. All of them were placed in Westergren sedimentation tubes (the last portion of blood in celloidin prepared ones). The sedimentation rate was read at suitable time intervals during 70-90 minutes. The bloods tested, even including bloods with reversed SR, disclosed good agree- ments without significant differences. From this it may be concluded that the ac- tual anticoagulants in the small amounts employed do not alter the rate of sett- ling inherent in native blood (celloidin tubes). Nor did the doubling of the pre- scribed amount of oxalate (Heller and Paul 1. 0.) delay the settling rate in whole blood or in diluted blood. As the sedimentation rate of oxalated blood is thus practically identical with that of native blood, oxalated blood has been preferred in the following for technical reasons. Factors other than a slight increase in elec- trolyte content must thus determine the peculiar curve shape and be the cause of reversed SR.

In addition to the effect of electrolytes on SR a special problem may be touched upon in this connection. HorBnyi attributed reversed SR to wine Citratwirkung. To settle this question we carried out serial comparative studies between SR of oxalated bloods diluted respectively with 3.0 per cent citrate solution and with 0.3-0.9 per cent saline solutions, employing 4 parts of blood and 1 part of the diluting agent. Slowly as well as rapidly settling bloods were examined. Good agreements with SRocB were obtained when 0.5-0.7 per cent salines were used. Sligbtly increased values were encountered with 0.9 per cent and somewhat lower values with salines below 0.5 per cent. These findings, which agree well with analo- gous experiments on heparinized blood (Nielsen 1942), show that (except for a possible slight ionic action) there is no essential difference between dilution with citrate or saline.

168 E 11. MARTENSSON ABD II. A. HANSEN.

3. Plasma Proteins.

Plasma proteins play a central r81e in the production of red cell aggregates and are the main factor determining their sizes, thereby greatly affecting the sedimen- tation velocity. The total plasma protein concentration does not primarily decide the sedimentation rate, but this depends more on the relations of the various proteins to one another. As early as 1921 F l h r m s demonstrated the strongly accelerating effect of fibrinogen, which has been confirmed by several authors (Westergren 1924, Bendien and Snapper 1931, Wunderly and Wuhrmann 1944, Buckley, Powell and Gibson 1950, Hardwicke 1951, among others). The acceler- ating effect of globulins is less marked but may be evident (Malmros and Blix 1946), whereas albumin rather has a retarding influence. The correlation between the proteins mentioned and the sedimentation rate has been regarded as so close that several writers have given formulas for calculating the speed of fall, if protein fractions are known (Westergren, Theorell and Widstrom 1931, Bendien, Neuberg and Snapper 1932, Westergren and Stavenoi 1951). Gordon and Wardley (1943), however, have found that some special fractions can act on the sedimentation rate in a decisive way (euglobulin e. g. accelerates whereas globoglycoid and nucleo- protein inhibit - t h e sedimentation) and may- render hazmdous the*aledation be- fore mentioned.

The protein composition is of less importance in the present investigation, as the relations of the various fractions to one another are kept constant, and only the total plasma protein concentrations are changed in the dilution series. It may be questioned whether the aggregating effect of the different protein fractions is a linear function. Within limited ranges, e. g. whole blood and 80 per cent of blood, the ratio, however, may be considered linear, and it may be permissible to omit protein determinations. The influence of varying absolute protein concentrations will be discussed later on in conjunction with the influence of diminished red cell volume.

d Influenoe of Tube Width.

When examining bloods with high settling rates we observed in tubes con- taining samples of 100-90-(80) per cent of blood that the cellular column often developed a conspicuously granular appearance, and that some large red cell conglomerates sometimes remained in the plasma layer. Duplicates could also differ greatly from each other. This indicated a very pronounced tendency to aggregation, and there were reasons to suspect that the tube width was too narrow to allow the aggregates to fall freely, thereby possibly giving rise to reversed SR.

Employing the Westergren technic (80 per cent of blood and 20 per cent of citrate) Wiemer (1927) closely examined the effect of the tube width, and found that a diameter equal to or larger than 2.6 mm did not impede the sedimentation. Kolmer and Boerner (1947) giving an account of different SR methods, state a tube bore less than 2 mm to be insufficient to secure an even sedimentation. In a comparison between the SR of practically undiluted citrated blood and that of

STUDIES OX FACTORS INFLUESCIKQ ERYTHROCYTE PEDIMENTATION RATE. 169

E to .- c loo

2 ao

E

I

60

40

”, 20

.- - a E

v)

10 20 30 40 50 60 70 80 90 100 110 120 Time in minu tes

Fig. 3. Influence of varying tube width on the SR of whole blood. Readings made at different time intervals up to 2 hours (wedigrams,). The tube diameters are given in mm against each curve.

blood diluted 20 per cent with citrate, Westergren (1924, 1937) observed delayed settling in whole blood caused by intense aggregation and therefore recommended wider tubes when handling whole blood. This matter has also been realized by Kaulla (1939) and Ostner (1942).

In our experiments to determine the course of the dilution curves (Fig. 1) or- dinary Westergren tubes with an internal diameter of 2.5 mm were used. On ac- count of the information derived from the literature cited above, it was necessary to examine the influence exerted by different tube bores.

With the same oxalated blood sample (Hb 103 per cent according to Haldane’s standard, hematocrit 45 per cent), the sedimentation rates were recorded at suit- able intervals in selected tubes with varying bores on: 1) whole blood, 2) whole blood + citrate (4 : I), and whole blood diluted with i t R own plasma (4 : 1). The last-mentioned was chosen to get an idea of the significance of the red cell volume. The results are presented as )medigramso (sedimentation curves) in Fig. 3-5.

The curves consist of three phases: 1) the phase of aggregation including the first bend, 2) the period of rapid fall represented by the linear part, and 3) the phase of packing represented by the last bend of the curve.

In the first series (Fig. 3) representing undiluted blood the tube diameter ranged from 2.1-16.8 mm, the blood column being 200 mm. The curves obtained with the narrow tubes (including Westergren tubes) disclosed a retarded and uneven sedimentation. A tube width larger than 4 mm secured a maximal velocity during the period of rapid fall. In the present case with a high SR and a normal hema- tocrit value, however, a tube diameter of 5 mm also seemed to be required to prevent a delayed sedimentation due to a prolonged aggregation phase. Larger diameters (up to 16.8 mm) did not markedly shorten the aggregation period, and the sedigram did not differ much from that of the 5.0 mm tube. Smaller diameters may under certain conditions (high SR and hematocrit value) considerably pro- long the aggregation phase and give rise to uneven one hour readings. A tube

Fig. 4. Influenoe of varying tuhe width on the SR of blood diluted with oitrate (4 pclrts of whole blood and 1 part of citmte solution) - nsedigrsmsr. The tube diameters are given in mm against eech

ourve. The same blood as in Fig. 3.

bore of 5 mm should therefore be chosen to avoid irregularities when employing whole blood.

In the second series (Fig, 4), consist&g of the blood citrate mixture, the tube bore varied between 1.8-4.8 mm. In accordance with the results obtained by previous writers no marked deviations between the individual curves were noted, and the one hour readings coincide.

In the third series with oxalated whole blood diluted 4 : 1 with plasma the tube bores varied between 2.14.3 mm. The plasma was thus not diluted, but the red cell volume waa reduced to the same degree as in series 2.

As can be seen from Fig. 5 the shapes of the sedigrams varied markedly with the tube width. The rapid falling of the cell aggregates was inhibited strongly in

120

.c 100 3 80 c -2 60 2

40

g 20

Time in minutes Fig. 5. Influence of varying tube width on the SR of blood diluted with its own plasma (4 parts of oxdated whole blood and 1 part of plasma) - rsedigramsr. The tube diameters are given in mm

against each ourve. The same blood &B in Figs. 3 and 4.

STUDLE3 ON FACTORB INBLUENCINB ERYTHROCYTE SEDIMENTATION BATE. 17 1

the 2.1 mm tube, less in the 2.3 mm tube, and presumably not a t all in the 4.3 mm tube. Consequently the abnormalities observed with whole blood in n m o w tubes were not eliminated by the reduced red cell volume. It is further evident that the diminished hematocrit value shortens the aggregation time.

The findings concerning the tube width called for a reinvestigation of the dilu- tion curves for high SR and the phenomenon of reversed SR. Tubes with a bore of about 5 mm were employed for the highest blood concentrntions (more than 80 per cent of whole blood). The result of this reinvestigation was that the first rise of the curves became more or less levelled but never so much that the original shape was changed, and thus the SR values remained reversed. A reversed SR is also to be expected if the phase of cell packing occurs before the time of reading (one hour), since the higher the red cell volume, the smaller the maximal extent of fall.

6. Influence of Red Cell Packing. In the dilution curves shown in Fig. 1 the SR was read after one hour. As bas

just been stated, a narrow tube bore is not always sufficient to explain reversed SR. A contributing cause could be sought in the packing of erythrocytes. To eluci- date this question, the kinetics of the packing phase of the SR curve were studied in detail in a dilution series read at 15-minute intervals; 6 mm tubes as well as Westergren tubes were employed. The results are presented in Fig. 6.

After 15 minutes, in both types of tubes, the SR was lower in whole blood than in the subsequent dilutions, probably due to a prolonged aggregation phase of the more concentrated samples. After 30 minutes there was seen no reversed SR in the 5 mm tubes (whole blood via-8-vis 80 per cent of blood), but the dilution curve kept its convexity, although levelled. Corresponding readings in the Westergren tubes disclosed as usual a pronounced convexity with reversed SR. After 60 minutes an extreme packing had occurred in the tubes with the highest blood cell volumes, preventing further sedimentation, whereas sedimentation was progressing in the more diluted specimens. Reversed SR had reappeared even in the 5 mm tubes, and the familiar curve shape was thus re-established, During this interval of time the vertex was displaced to the right, i. e. towards increased dilution. A peculiar phenomenon, difficult t o explain, must be pointed out, namely, that narrow tubes will often disclose a certain promoting effect on the falling rate of the moderately diluted bloods - see Fig. 6.

Some writers report the SR value in per cent of maximal sedimentation cal- culated from the hematocrit value. Applying this procedure, however, we find that the dilution curve still keeps its original shape, probably because larger and looser aggregates are formed in the more concentrated blood samples, and maximal paoking will never occur.

To delay the onset of cell packing the blood column was lengthened to 450- 600 mm, the tube diameter being 5 mm.1 With this technic dilution curves were

1 The arrangement of Swedin (1. 0.) to prevent the packing of the red cells, with the lower end of the 8edimentat.ion tube plunging into blood or a blood citrate mixture in a small receptacle, wa8 tried but not found applicable to rapidly settling bloods.

12-531620. Acfa med. Scandinau. Vol . CXLVI .

172 E. H. M~RTEXSSON AND H. A. HANSEX.

% whole blood in blood citrde mixture Fig. 6. SR of different dilutions of whole blood ($dilution curves)) read after 15, 30 and 60 minutes using different types of tubes. x- x = Westergren tubes, 2.5 mm in bore; X . G . = tubes 6 mm in bore, the @falling distancer in both cases being 200 mm. - The upper curve (0-0) shows

SR obtained after 60 minutes with tubes 5 mm in bore and with a ofalling distancen of 500 mm.

i " ' " ' " " " ~ ' 0 3 0 4 0 50 60 /5 90 Time in minutes

Fig. 7. Influence of tube width and tube length on SR in whole blood end in different dilutions. Road ings made at different time intervals - nsedigrams,. 0-0 = Tube diameter 2.5 mm, nfding diatanceu 200 mm (Westergren tubes). X - - - - - . - x = Tube diameter 5 mm, nfalling dis- tance, 500 mm. The percentage of blood in the blood citrate mixtures is given against each curve.

STUDIES ON FACTORS INFLUENCING ERYTHROCYTE EEDIMENTATION RATE. 173

performed on the same blood as that used in Fig. 6. The results are represented by the broken line a t the top of Fig. 6 and show a steady, even drop with in- creasing dilution as typically found with moderately settling bloods.

The reversed SR for rapidly settling bloods has thus been cancelled. Sedigrams were also recorded with the last-mentioned long tubes as well as the

Westergren tubes. Whole blood and blood citrate mixtures of 80 and 70 per cent were tested. The data are presented in Fig. 7.

As can be seen the packing effect has been eliminated during the first hour in the long tubes. Although exhibiting a prolonged aggregation period the sedi- gram of whole blood, due to its greatest velocity during the phase of rapid fall, will intersect the other curves, and after one hour disclose the highest value. The sedigrams of the Westergren tubes, however, are typically reversed agreeing with HorBnyi’s curves. Regarding sedimentation in 80 per cent of blood in the 5 mm tubes as compared with the Westergren tubes a parallel course is found during the period of rapid fall. This indicates that settling - read per unit of time on this linear part - will coincide and best express the aggregation state and the true falling speed. This is also valid for whole blood provided that the tube bore is adequate. The same manner i f taking readings has been proposed by Lundgren (1927) and Rourke and Ernstene (1930), but being laborious i t has not been used in practice.

From these experiments the following general conclusions can be drawn: 1) that the true sedimentation rate in whole blood is always greater than in any dilution, and 2) that the reversed SR can be regarded as an artefact mainly due to too narrow and too small sedimentation tubes.

6. Influence of Red Cell Volume and Plasma Dilution on the Dilution Curve.

It has already been possible to explain the reversed SR and a t the same time also the factors deciding the ascending branch of the dilution curve. The descending branch, however, has not been discussed. Since the performance of a dilution series implies a dilution both of blood corpuscles and of plasma, the factors gover- ning the descending branch must be sought in both of these variables. To elucidate the r61e played by each of these factors the following experiments were carried out on slowly, moderately and rapidly settling bloods.

80 ml oxalated blood was taken through venipuncture and the hematocrit value determined. The effect of varying amounts of red cells (65 t o 20 per cent) was investigated in whole blood as well as in certain mixtures of whole blood and citrate solution. In each such series the erythrocytes were concentrated by pipetting off plasma or plasma-citrate mixture after sedimentation or cautious centrifugation (2,000 r. p. m.). The dilution was accomplished by adding plasma or plasma citrate mixture, the protein concentration thus being kept constant in each series. For each specimen the red cell volume and for each series the plasma concentration were calculated from the hematocrit value obtained with whole blood. All the specimens were placed in sedimentation tubes (blood column 200 mm). For rapidly settling bloods and for erythrocyte volumes of more than 35 per cent a tube width

174 E H. MARTENSSON AND H. A. HANEEN.

Hemotocrit value

Fig. 8. The relationship between SR and hematoorit value when different amounts of blood cells are suspended in undiluted plasma and in different plasma citrate mixtures. The diagram - broken line - shows the separate effects of plasma dilution (a) and of reduced erythrocyte volume (b) as well as the resultant effect (c) due to the Westergren dilution technic. The plasma contents are given against

each curve. Normally settling blood.

of 5 mm was used. The sedimentation rates were read after one hour. The results are given as a mean of two or more determinations.

In Fig. 8 the data from a case with lzormal SR are presented (hemoglobin 102 per cent and hematocrit 44.6 per cent). At such a low speed of fall there can be no packing effect. The SR values are plotted against the ordinate and the volume of packed erythrocytes against the abscissa. Sedimentation rates a t varying erythrocyte volumes in the same fluid medium (the same protein content) are linked to form curves. The percentage of plasma in each series is given beside each curve in the Figures.

From Fig. 8 it is seen that the sedimentation is practically arrested a t high erythrocyte volumes. When the red cells decrease the sedimentation velocity in- creases a t first slowly, later on rapidly and proportionally to the reduced red cell volume. At very low erythrocyte concentrations the readings are difficult and somewhat arbitrary due to the development of indistinct borderlines. The read- ings have always been made a t the level where the erythrocytes fill out the lumen of the tube, and a dense corpuscular column becomes prominent. This means that the SR values obtained are somewhat too high. SR values for specimens with a hematocrit value lower than 20 per cent are not given, since they could not be read with any degree of accuracy.

The curves give a good illustration of the accelerating effect of a reduced red cell volume. This effeot is greater in undiluted plasma than in any of the plasma dilutions. Nygaard, Wilder and Berkaon (1936) studying the correlation between packed red cell volume and blood viscosity found a linear ratio for erythrocyte

fiTUnlEd OY FACTORS INFLUEUCIKQ ERYTHROCYTE EEDZMENTATlON RATE. 155

Fig. 9. The relationship between SR and the relative plasma contents (undiluted plasma = 100 per cent) in the plasma citrate mixtures at different levels of erg- throcyte volume, which are given against each curve. The intersecting line shows the effect on SR when Westergren’s dilution technic is employed, and the red oells vary between 25-50 per cent in whole blood corresponding to 2 0 4 per cent in the Westergren

dilution mixture. The same blood 8 s in Fig. 8.

.s 25 0 Ic.

E E

v)

.L1

3 1

% plasma in plasma citrata mixture

volumes between 15-60 per cent. Blood viscosity also rapidly increased with further corpuscular concentration. These findings agree well with the curves pre- sented here. In Fig. 8 a *triangulan, diagram is inserted to demonstrate the combined effect

of plasma dilution and reduced red cell volume when whole blood is diluted with citrate in proportions 4 : 1. SR of whole blood was found to be 7 mm, by mere plasma dilution SR was reduced t o 1 mm, and by simultaneous reduct,ion of the red blood corpuscles a SR,, value of 4 mm was obtained. The side of the diagram designated *a$ shows the delaying effect of plasma dilution. Side nb, denotes the accelerating effect of diminished red cell volume (effect of anemia). As can be seen the effect of plasma dilution exceeds the effect of red cell reduction. The resulting total dilution effect)), side UCD in the diagram, brings about a decreased settling rate in accordance with the observations on the dilution curvea (Fig. 1) for slowly and moderately settling bloods.

In Fig. 9 the significance of altered plasma concentration is illustrated, the erythrocyte concentration being kept constant. The data refer to the same trial as in Fig. 8, but are arranged differently. The SR values are plotted against the ordinate, and the percentages of plasma in the plasma citrate mixtures are plotted against the abscissa. The curves combine SR values with the same erythrocyte concentrations, which are given beside the respective curves, 40-20 per cent. The curves exhibit a somewhat bent shape, indicating that there is no direct rela- tion between the SR values and the plasma dilutions, in this respect differing from the ratio between SR and erythrocyte concentrations within the same range 8s shown in Fig. 8.

The isolated effect of either altered erythrocyte concentration or plasma dilu- tion has just been considered. When Westergren’s technic is used, however, both of these factors are changed simultaneously. It must be kept in mind, when diluting whole blood, that red cells and plasma are mostly diluted to different degrees since they seldom occur in equal proportions. Thus the higher the erythrocyte concentration, the greater the plasma dilution and vice versa.

176 E. II. MARTENSSON AND H. A. IIANPEN.

110

loo e 9 0 -

6 80-

- -

L 7

"O 12k 20%

Hematocrit value Fig. 10.

% plasma in plasma citrate mixture Fig. 11.

Fig. 10. Similar experiment to that in Fig. 8 on a moderately settling blood. For further explanations, see the text to Fig, 8. - Big. 11. Experimental data, arranged similarly to that in Fig. 9, on the

mme moderbtely settling blood as in Fig. 10. For further explanations, Bee the text to Fig. 9.

To find out the range of variation of SR in relation to different hematocrit values when WeBterqren's technic is applied, the following procedure was practised. Provided the erythrocyte concentration in whole blood is known, the red cell volume as well as the plasma concentration in the plasma citrate mixture can be calculated. For instance, a blood whose hematoorit value is 60 per cent gives - with a dilution of 4 : 1 - 40 per cent in the dilution mixture, and the plasma citrate mixture will contain 67.7 per cent of plasma. Correspondingly 25 per cent hematocrit gives 20 and 75 per cent respectively. If the values for the plasma concentrations are plotted against the corresponding erythrocyte volumes, the SR,, can be obtained from Fig. 9. If these values are joined, the crossing curve is obtained, giving the possible range of variation of the Westergren SR when erythrocyte volume in whole blood varies between 60-25 per cent. Thus in the present trial - simulating anemia - the SRcs increased from 1 to 17 mm in one hour.

Fig. 10 and 11 show curves similar to those in Fig. 8 and 9 for a moderateEy settling blood (SR,, 21 mm, hemoglobin 99 per cent and hematocrit 41 per cent). They do not differ essentially from those disclosing a normal sedimentation rate. It is, however, evident that the effect of plasma dilution is much more prominent

STUDIES ON FACTORS LNFLUENCINQ ERYTHROCYTE SEDIMENTATION RATE. 177

Hematocrit value X plasma in plasma citrate mixture

Fig. 12. Fig. 13. Fig. 12. Similar experiment to that in Fig. 8 and 10 on a rapidly settling blood. For further explana- tions, see the text to Fig. 8. - Fig. 13. Experimental data, arranged similarly to that in Fig. 9 and 11, on the same rapidly settling blood aa in Fig. 12. For further explanations, see the text to Fig. 9.

than for a normal SR, and greatly exceeds the likewise increased effect of reduced red cell volume. From Fig. 11 it can also be seen that the variations in SR,, caused by different erythrocyte volumes are much greater than in the former example (increasing from 8 mm for 40 per cent red cell volume to 56 mm for 20 per cent cell volume).

In Fig. 12 and 13 are shown corresponding curves from an experiment on a rapid2y settling blood (SRcB 87 mm, hemoglobin 100 per cent and hematocrit 43.5 per cent). The curves in Fig. 12 for the more concentrated blood specimens exhibit it distinct inflection at about 40 per cent erythrocytes, probably due to the onset of red cell packing. The curves representing undiluted and 83.6 per cent plasma intersect. This is certainly due to a prolonged aggregation time in undiluted plasma at high erythrocyte concentrations, which combination may sometimes give rise to reversed SR even at relatively slow speeds obtained by the Westergren technic.

From the inserted triangular diagram it is seen that the plasma dilution effect (a) is less than the cell dilution (anemia) effect (b). At still higher SR,, the paoking of erythrocytes will make this difference more evident. The net effect (c) results in a higher SR value in the diluted blood and thus causes reversed SR in accord- ance with the dilution curves for rapidly settling bloods in Fig. 1.

In Pig. 12 is also shown the influence of red cell volume at low plasma con-

178 E. u. M~RTESSSOB AND H. A. HANSEN.

tents (15.8 per cent). Here it is evident that the effect of plasma dilution exceeds by far that of anemia, resulting in strongly decreased SR values in agreement with the dilution curves (Fig. 1).

The plasma dilution curves in Fig. 13 differ somewhat from those formerly presented in having a more levelled course for higher plasma contents. This is due mainly to the effect of cell packing. A contributory cause may also be that the sizes of the aggregates are only slightly affected by a moderate plasma dilution, if a high aggregation tendency exists. In Fig. 13 is further shown the possible variation in SRcB, when erythrocyte concentration varies from 40-20 per cent (50-25 vol. per cent in whole blood) resulting in an increase in SR,, from 63 to 144 mm respectively.

The experience gained from these experiments can be summarized as follows: 1) reduced red cell volume causes accelerated sedimentation rate in undiluted aa well as diluted plasma (Fig. 8, 10 and 12), 2) for each erythrocyte volume the sedi- mentation rate decreases with increasing plasma dilution (Fig. 9, 11 and 13), and 3) the latter effect exceeds the former if no irregularities (prolonged aggregation time) or obstacles (red cell packing) are present.

When diluting whole blood (Fig. 1) the citrate will mix only with the plasma, and the plasma protein8 become relatively more diluted than the erythrocytes. This contributes to make the effect of plasma dilution predominate over that of the reduced red cell volume, resulting in a steady even drop of the SR values on successive dilution. The same conclusion is also valid for the descending part of the dilution curve for a rapidly settling blood, and if proper conditions are present (with respect to tube width and height of fall) the dilution curve for a rapidly settling blood will throughout exhibit a continuously falling course with increased dilution, and thus the ascending branch as well as the reversed SR is cancelled.

Discussion. BRevereed SR,.

In the above investigations the authors have analyzed the variation of sedimen- tation rates in whole blood diluted with different amounts of iso-osmotic citrate solution (dilution curves, Fig. 1). In slowly and moderately settling bloods SR wae found to decrease with increasing dilution. But with rapidly settling bloods, the problem was encountered of meversed SRn, i. e. that the SR of 80 per cent blood mixture exceeded that of whole blood. This problem was especially studied.

& h e r (1942) and Hortlnyi (1943) have closely dealt with the same phenom- enon. Ostner’s material differs from the present authors’ and other writers’ in disclosing reversed SR not only for rapidly settling bloods but also for compar- atively slowly settling bloods (Fig. 2). Ostner has, however, realized the im- portance of a wider tube bore especially with a high erythrocyte volume and a pro- nounced aggregation tendency.

Hortlnyi has carried out fibrinogen, albumin, and globulin determinations and found that, if protein composition were normal or if globulin or fibrinogen respec-

6TUDIEB ON FACTORS INFLUENCINff ERYTHROCYTE SEDIMENTATIOS RATE. 179

tively were increased, the sedimentation velocity was greater in native blood (celloidin tubes) than in citrated blood (Westergren technic), while, on the con- trary, if both globulin and fibrinogen were increased the rates became reversed. HorBnyi attributed this phenomenon to neine Citratwirkungs. It was concluded that citrate will sometimes retard and sometimes accelerate the velocity of fall, depending on the protein composition. Horsinyi’s interpretation must be rejected, since by changing the experimental conditions it has been possible in the present investigation to cancel the reversed SR and thus show that dilution of whole blood with citrate solution always results in diminished SR values. Thus Horinyi has disregarded such factors as the influence of the width and the length of the tube, the effect of red cell packing, and the dilution of blood corpuscles and of plasma, which factors may be considered sufficient to explain reversed SR and, a t the same time, also the ascending branch of the dilution curve for rapidly settling bloods. As to the tube width Horinyi has used ordinary Westergren tubes. From the experiments shown in Fig. 3 and 5 it appears that this tube bore is too narrow to allow free falling of the large aggregates in whole blood in all instances. Even with an adequate tube bore (4-5 mm) reversed SR nevertheless often occurs. The reversal is especially marked when there is a very high sedimentation tendency. Here it must be emphasized again that a prolonged aggregation time may perceptibly influence the one hour readings too, and that aggregation time in its turn is dependent on both red cell volume and tube width (thus increased erythrocyte volume and/or narrow tube bore will lengthen the aggregation phase and vice versa).

Another factor of importance for reversed SR is to be sought in a too short falling distance for the red cells, increasing the effect of cell packing. This factor is more pronounced the higher the red cell volume. Although it may be evident from a comparison between the curves in Fig. 3 and 4 that SR,, is greater than SR,, during the period of rapid fall, it has been possible to prove that this is really the case in the experiments in Fig. 6 and 7, where the effect of cell packing has been eliminated by increasing the height of fall (one hour readings). Thus if an adequate tube bore and an adequate height of fall are chosen, it can be proved that the velocity of fall is always greater in whole blood than in any dilution with citrate. A specific oeffect of citrate,, in the sense Horhy i gives to this expression, seems not justified. Another strong support for the assertion made is that the dilution of oxalated whole blood with sodium chloride or citrate gives the same results. Nor does addition of solid citrate to whole blood alter the sedimentation rate under correct experimental conditions,

From the data presented in Fig. 8 and 10 appears that the effect of diluting the plasma much exceeds that for the red corpuscles (anemia effect), resulting in a delayed sedimentation rate.

This is also the explanation of the dilution curves for slowly and moderately settling bloods and of the descending branch for rapidly settling bloods. The ))citrate effect)) is thus mainly due to the dilution of plasma.

180 E. H. MARTEMSON AND H. A. HANSEN.

Polycgthemia and Anemia.

From a clinical point of view polycythemia and anemia are of special interest in connection with evaluation of the SR. From the experiments presented, it may be deduced that no sedimentation occurs with a high red cell volume, i. e. with polycythemia. Thus the SR procedure is of no clinical value when the hematorcrit exceeds 55 per cent. Under such oircumstances (hemoglobin more than 120 per cent) the erythrocyte concentration has to be corrected to a normal value (hema- tocrit 43 per cent, or hemoglobin 100 per cent). When blood for the sedimentation tests is collected as whole blood it will be an easy matter. The whole blood must be diluted with its own plasma, and the amount of plasma to be added, X i d ,

a - 100 100 can be calculated from the following formula: X = ___ x M, where a denotes

the hemoglobin value in per cent, and M the amount of whole blood to be diluted. In the literature the discussion on the effect of anemia has been rather conflicting.

From our trials it is clear that reduced red cell volume will always result in an increased sedimentation rate provided the plasma concentration is kept constant. Such experiments are presented in Fig. 8,10, and 12. The curves are approximately straight lines for erythrocyte concentrations of 40-20 per cent. If a dilution tech- nic, for instance Westergren's, is employed, the composition of the suspension medium will vary with the erythrocyte volume: the more reduced the red cell volume, the less the degree of the plasma dilution. This results in a somewhat bent course of the curves, as appears from Fig. 9, and 11 (intersecting curves). If anemia is imitated by reducing red cells by addition of plasma and SR, , is deter- mined, the sedimentation rates will increase in a regular way with decreasing cell volume. I n the case of mormab SROB (less than 7 mm in one hour, hemoglobin 100 per cent, hematocrit 43 per cent), the influence of diminished red cells is com- paratively slight (Fig. 9), but with high SRcB this influence is considerable (Fig. 11). At very high sedimentation rates, however, the effect of anemia again becomes much smaller, and the increase in sedimentation rate may sometimes correspond only to the decrease in packed red cells (Fig. 13).

It has been proposed by several authors (Gram 1928, Rourke and Ernstene 1929, Jersild 1934, Wintrobe and Landsberg 1935) to correct the SR for the degree of anemia to normal hematocrit or hemoglobin values, while others (Wecltergren 1924, Lebel and Lottrup 1933, Dahl 1944, Gjerdsjo 1945) have considered this less important. As the effect of anemia - according to our findings - can be con- siderable, it would seem desirable to present a correotion table for different degrees of anemia. From a practical point of view, however, it aeems sufficient to know the mpper normal limitu a t different hemoglobin values. If the ,upper normal limito for SR,, a t 100 per cent hemoglobin is established as 7 mm, SR,, a t 120 per cent hemoglobin will be 2 mm, a t 80 per cent 12 mm, a t 60 per cent 18 mm, and a t 40 per cent 25 mm in one hour. As has been mentioned before and has also been stated by Gram (1. c . ) , and by Rourke and Ernstene (1. c.)> it is obvious that the SR values a t any height vary regularly with the red cell volume, and since the

STUDIES ON FACTORS INPLUENCINQ ERYTHROCYTE SEDIMENTATION RATE. 181

above given limits a t different hemoglobin values always correspond to a normal value of 7 mm a t 100 per cent hemoglobin, the correction of SR values below these mormal limits)) will be of no interest. If in anemia the SR values exceed the limits stated, it is necessary to search for some cause of the increase other than a simple reduction in the red cell volume: in the first place changes in the plasma proteins produced by the disease. This view seems to agree well with Gjerdsjo’s opinion, that idiopathic hypochromic anemia as well as uncomplicated posthemorrhagic anemia exert little effect on the SR. In the literature there are repeatedly reported cases with pronounced anemia exhibiting extremely low SR. This is comprehen- sible from the present experiments. Thus bloods disclosing SR,, of 0-1 mm in one hour at 100 per cent hemoglobin will hardly increase their sedimentation veloc- ity when the red cells are diminished.

Possibly the above established mpper normal limits) at different hemoglobin values are fixed too high for hypochromic anemias and somewhat too low for hyperchromic anemias. The limit values are derived from bloods exhibiting prac- tically normal colour indexes. According to several investigators (Oestreich 1931, Bendien, Neuberg and Snapper 1932, Lindeboom 1934) the erythrocytes in hypo- chromic anemias often disclose a diminished specific gravity, which can influence the sedimentation rate. According to Stokes’ formula, which has been widely applied in discussions of sedimentation problems, the force causing the erythro- cytes to fall is proportional to the difference between the specific gravities of erythrocytes and plasma. Since the specific gravity of the plasma is practically constant, that of the erythrocytes is the variable of importance for the SR. A diminished specific gravity of the red cells will thus cause a retarded settling rate. An increased specific gravity of the erythrocytes (mainly in pernicious anemia) will, on the contrary, hasten the sedimentation. The increase in the specific gravity of red cells is, however, less pronounced than the decrease which can occur, but possibly should also be taken into consideration. No precise evaluation of the importance of the altered specific gravity of the erythrocytes in anemias can be made from the present investigation, but the authors will call attention t o the fact that Bendien, Neuberg and Snapper (1. c.), in their formula for calculation of SR, have inserted a correction for variations in the specific gravity of red cells in anemias using the colour index. In this way they obtained good agreements between calculated and experimental values. Within moderate deviations of the colour index, however, the ))upper normal limits)) given above may serve as a practical guide.

A reduced ability to aggregate has been attributed especially to young erythro- cytes (reticulocytes), causing a low SR in anemia (Westergren 1924, Aberg 1942 among others). It is doubtful whether these are present in a simple anemia to such an extent as to exert any determining effect on SR, and this has not been convincingly verified by exchange experiments on blood cells. Exchange experi- ments have also been carried out by us, but we never succeeded in obtaining the expected changes. In exchange experiments, however, it is difficult to free the blood corpuscles from their own plasma, and, besides, damaged cells and injuries to cell membranes will be encountered. From what has been said above it is evident

182 E. H. MdRTENBSOH AND 11. A. HANBEX,

that i t is not necessary to consider the presence of immature red cells to explain a slow sedimentation rate in extreme anemia. It cannot be denied, however, that reticulocytes and bizarrely shaped red cells may contribute to a low SR.

If a correction for the influence of anemia is nevertheless desired, this can be performed according to the formula and the procedure described for the correc- tion of polycythemia, except that X now denotes the amount of plasma to be removed.

Whole Blood or Diluted Blood for the Sedimentation Tests?

It has been discussed whether whole blood or diluted blood is to he preferred in sedimentation tests. A better differentiation between normal and pathological conditions has been attributed to the SR of whole blood, but the settling is more capricious and gives rise to greater deviations than in diluted blood. When whole blood is chosen a tube width of 5 mrn must be used to secure free falling and a constant aggregation time, since in whole blood this time is often prolonged to a variable extent in narrow tubes, a factor which may influence the one hour readings. To make a differentiation of higher SR values possible, it is necessary to employ a tube length with a falling distance exceeding that of the Westergren tubes.

In the Westergren dilution procedure the velocity of fall is decreased as com- pared with that in the whole blood procedure. This brings about a good differen- tiation also for rapidly settling bloods. Furthermore, the aggregation phase is more constant, and the standard error less than that of the whole blood test. A tube width of 2.5 mm is also sufficient to allow the aggregates to fall freely. The amount of blood is thus much smaller than that required for whole blood tests. For clinical routine work the dilution technic of Westergren must therefore be regarded as the most convenient. .

Su iamnry.

The influence on the erythrocyte sedimentation rate (SR) of dilution of whole blood with varying amounts of iso-osmotic citrate solution has been investigated, and so-called dilution curves of two main types are presented. One type shows a steady drop with increasing dilution, the other a t first shows increasing SR values, reaching a maximum, and then decreasing values. The latter curve type pre- dominates in bloods with a high sedimentation rate. The causes of the curve shapes and especially the ureversed SR)), i. e. a higher SR in blood diluted 4 : 1 than in undiluted blood, have been analyzed and explained.

The influence of tube width and length, of cell packing and of aggregation time, has been experimentally studied. If a sufficiently large tube bore and an adequate tube length are chosen, it is possible to eliminate reversed SR, and thus the real sedimentation speed has always been found greater in whole blood than in any dilution. A dilution procedure according to Westergren was nevertheless found to introduce fewer errors and be the most convenient for clinical work.

The influences of changes in the erythrocyte volume and of the plasma dilution

6TUDIE3 ON FACTORS INILUENCINO ERYTEROCYTE BEDIMENTATION RATE. 1 83

have likewise been studied concomitantly as well as separately. The retarding effect of plasma dilution exceeds the accelerating effect of reduced red cell volume and explains the successive decrease in SR on dilution.

Some points of view concerning polycythemia and anemia in relation to sedi- mentation rate are given. In polycythemia dilution with plasma to normal hemo- globin value is recommended. In anemia the ,upper normal limits)) for SR at dif- ferent hemoglobin values are given. This is considered useful and more convenient for clinical routine work than any correction procedure.

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