OBSERVATIONS ON THE DEVELOPMENT OF THE HIGH BLOOD SEDIMENTATION RATE IN RHEUMATIC CARDITISi By ALVIN F. COBURN AND E. M. KAPP (From the Department of Medicine, College of Physicians and Surgeons, Columbia University, and the Presbyterian Hospital, New York City) (Received for publication June 18, 1936) The practical value of serial determinations of the erythrocyte sedimentation rate as an aid in detecting the presence of rheumatic activity has proved itself thoroughly among clinicians. Our findings during the last five years have been in complete accord with those of a number of other investigators (1 to 11, inclusive). The erythro- cyte sedimentation rate is regularly elevated in patients with rheumatic carditis, and even minor fluctuations seem to be directly related to the clinical course of the disease. Except when as- sociated with congestive heart failure, a decreas- ing sedimentation rate nearly always reflects di- minishing activity of the rheumatic process. The present paper deals with the initial development of a rise in sedimentation rate at the onset of the rheumatic attack, and with an investigation of the factors in blood directly responsible for the change. The erythrocyte sedimentation rate in pharyngitis, scarlet fever and rheumatism As shown in a previous publication (12), an attack of acute rheumatism is usually preceded by an upper respiratory infection with hemolytic streptococcus, which we designate as Phase I of 1 The work reported in this communication was carried out under The W. K. Kellogg Foundation Fund. . 1. 60 40 20 60 40 20 the rheumatic attack. Following recovery from the primary infection, there is a symptom-free in- terval of from one to three (rarely five) weeks in length, during which the patient appears to be in perfect health. This we designate as Phase II. The rheumatic attack proper is referred to as Phase III. It has been possible in a number of known rheumatic subjects who have been under close observation for a period of years to follow the sedimentation rate from the onset of hemolytic streptococcus pharyngitis through Phase II into Phase III or complete recovery as the case might be. Many of these patients de- veloped typical acute rheumatism. Some escaped all signs of rheumatic recrudescence. The sedi- mentation curves were quite different, depending on whether the patient developed a rheumatic attack or not. Sample curves of both types are presented in Figures 1 and 2. It will be seen from these curves that the sedi- mentation rate was slightly or moderately ele- vated during pharyngitis in some of the individ- uals studied, and remained at a normal level in the others, irrespective of whether the infection was followed by rheumatism or not. In patients who escaped recrudescences (Figure 1) the sedi- mentation rate remained at its normal level or returned to normal in about two to three weeks 02030 40203 0 10 20 30 10 20 30 10 20 30 10 20 DAYS AFTER ONSET OF RESPIRATOQY INFECTION FIG. 1. SEDIMENTATION RATES OF RHEUMATIC SUBJECTS WITH HEMOLYTIC STRIWocOCCUS PHARYNGITIS WHICH WAS NOT FoLLoww BY RHEUMATIC SYMPTOMS. 715 4 a I 1 -11 I a 9 40 il t . a I
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OBSERVATIONS ON THE DEVELOPMENT OF THE HIGH BLOODSEDIMENTATION RATE IN RHEUMATIC CARDITISi
By ALVIN F. COBURN AND E. M. KAPP(From the Department of Medicine, College of Physicians and Surgeons, Columbia University,
and the Presbyterian Hospital, New York City)
(Received for publication June 18, 1936)
The practical value of serial determinations ofthe erythrocyte sedimentation rate as an aid indetecting the presence of rheumatic activity hasproved itself thoroughly among clinicians. Ourfindings during the last five years have been incomplete accord with those of a number of otherinvestigators (1 to 11, inclusive). The erythro-cyte sedimentation rate is regularly elevated inpatients with rheumatic carditis, and even minorfluctuations seem to be directly related to theclinical course of the disease. Except when as-sociated with congestive heart failure, a decreas-ing sedimentation rate nearly always reflects di-minishing activity of the rheumatic process. Thepresent paper deals with the initial developmentof a rise in sedimentation rate at the onset of therheumatic attack, and with an investigation ofthe factors in blood directly responsible for thechange.
The erythrocyte sedimentation rate in pharyngitis,scarlet fever and rheumatism
As shown in a previous publication (12), anattack of acute rheumatism is usually preceded byan upper respiratory infection with hemolyticstreptococcus, which we designate as Phase I of
1 The work reported in this communication was carriedout under The W. K. Kellogg Foundation Fund.
. 1.
60
40
20
60
40
20
the rheumatic attack. Following recovery fromthe primary infection, there is a symptom-free in-terval of from one to three (rarely five) weeksin length, during which the patient appears to bein perfect health. This we designate as PhaseII. The rheumatic attack proper is referred toas Phase III. It has been possible in a numberof known rheumatic subjects who have beenunder close observation for a period of years tofollow the sedimentation rate from the onsetof hemolytic streptococcus pharyngitis throughPhase II into Phase III or complete recovery asthe case might be. Many of these patients de-veloped typical acute rheumatism. Some escapedall signs of rheumatic recrudescence. The sedi-mentation curves were quite different, dependingon whether the patient developed a rheumaticattack or not. Sample curves of both types arepresented in Figures 1 and 2.
It will be seen from these curves that the sedi-mentation rate was slightly or moderately ele-vated during pharyngitis in some of the individ-uals studied, and remained at a normal level inthe others, irrespective of whether the infectionwas followed by rheumatism or not. In patientswho escaped recrudescences (Figure 1) the sedi-mentation rate remained at its normal level orreturned to normal in about two to three weeks
02030 40203 0 10 20 30 10 20 30 10 20 30 10 20DAYS AFTER ONSET OF RESPIRATOQY INFECTION
FIG. 1. SEDIMENTATION RATES OF RHEUMATIC SUBJECTS WITH HEMOLYTIC STRIWocOCCUS PHARYNGITIS WHICHWAS NOT FoLLoww BY RHEUMATIC SYMPTOMS.
FIG. 2. SEDIMENTATION RATES OF RHEUMATIC SUBJECrS WITH HEMOLYTIC STREPTOCOCCUS PHARYNGITIS WHICHWAS FOLLOWED BY SEVERE RHEUMATIC CARDITIS (ONSET OF RHEUMATIC ATTACK INDICATED BY ARROWS).
from the onset of infection. In patients who de-veloped rheumatic attacks, however (Figure 2),the subsidence of sedimentation rate after recov-ery from pharyngitis was interrupted by a secondsharp increase coincident with or just precedingthe onset of rheumatic symptoms.A similar study was made of a control group
of subjects with hemolytic streptococcus pharyn-geal infections who had no history of previousrheumatic disease. This group consisted of (a)15 young adults, mostly nurses, with acute pharyn-gitis, whose throat flora contained hemolyticstreptococcus in predominance, and (b) 10 pa-tients, mostly children, admitted to Willard
Parker Hospital with typical scarlet fever. Thesedimentation rate curves are presented in Fig-ure 3.
Eighteen of these patients recovered withoutdeveloping complications. Their sedimentationrate curves are indistinguishable from thoseshown in Figure 1. Five patients with pharyn-gitis and two with scarlet fever did develop com-plications during convalescence. All of thesecomplications were accompanied by renewed in-creases in the sedimentation rate. These second-ary increases were moderate, with the exceptionof one patient who developed, successively, otitis.mastoiditis, mild polyarthritis and electrocardio-
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BLOOD SEDIMENTATION RATE IN RHEUMATIC CARDITIS
>40 - CERVICALl~~~~~~~~~~~~~~~~ADEM1ITIS
440 CERVICAL ADErTIST F 44 45 46 47
20 -
10 20 30 10 20 30 10 20 30 10 20 30 10 20 30DAYS AFTER ONSET OF RESPIRATORY IiFECTIOM
FIG. 3. SEDIMENTATION RATES OF CONTROL SUBJECTS WITH HEMOLYTIC STREPTOCOCCUS RESPIRATORY INFECTIONS-PHARYNGITIS AND SCARLET FEVER.
graphic changes. In this case (Curve 35) thesedimentation rate rose to a level as high as thatwhich usually accompanies rheumatic carditis(cf. Figure 2). Similar observations have beenreported by Rhodin (13) who found a moderateincrease in sedimentation rate during scarlet feverfollowed by a second larger increase in the eventof complications, with a well marked minimumbetween them. The curves of several patientswhose sedimentation rates were determined atfrequent intervals showed cyclic fluctuations dur-ing the first week or two after infection similarto those observed by Rhodin (13) in scarlet fever.In our series of patients, this type of curve oc-
curred not only during convalescence from scarletfever (Patients 38, 39, 40, 41) but also duringPhase II of acute rheumatism (Patients 13, 21).
In summary, increases in sedimentation rate
may or may not occur during initial hemolyticstreptococcus pharyngitis. It does occur duringseptic complications and during sequelae character-ized by sterile inflammation.
Plasma proteins in relation to the erythrocytesedimentation rate
The literature dealing with the factors in bloodresponsible for an increased sedimentation ratecontains a wide variety of conclusions. How-ever, the majority of investigators are agreed thatthe decisive factors reside in the plasma or serum
rather than in the cells, and that abnormal sedi-mentation is accompanied by quantitative altera-tions in the protein fractions of plasma. Fahraeus(14) pointed out that the amount of fibrinogen or
serum globulin paralleled the sedimentation rate.This has been confirmed by Westergren et al.
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ALVIN F. COBURN AND E. M. KAPP
TABLE I
Serum protein values of nine patients with acute rheumatism, and of nine healthy control subjects
Patients with acute rheumatism Controls
Total Serum Serum Total Serum Serumserum albumin globulin A/G ratio serum albumin globulin A/G ratioprotein protein
grams per 100 cc. grams per 100 cc. grams per 100 cc. grams per 100 cc. grams per 100 cc. grams Per 100 cc.6.8 3.9 2.9 1.34 6.7 4.6 2.1 2.197.4 4.2 3.1 1.35 7.3 4.8 2.5 1.927.0 4.5 2.5 1.82 6.9 4.6 2.3 2.007.2 4.8 2.5 1.92 7.2 4.9 2.3 2.138.1 4.7 3.4 1.40 7.2 4.8 2.4 2.008.8 4.7 4.1 1.14 7.3 4.9 2.4 2.048.0 4.1 3.9 1.05 7.0 4.7 2.3 2.048.2 4.5 3.7 1.16 7.1 5.2 1.9 2.747.9 4.5 3.4 1.32 7.3 5.1 2.2 2.32
Average7.7 4.4 3.3 1.39 7.1 4.8 2.3 2.15
(15) and by Bendien and Snapper (16) for a
number of miscellaneous diseases. Gilligan andErnstene (17) found a striking correlation be-tween the quantity of plasma fibrinogen and thesedimentation rate in rheumatic fever. Highlevels of fibrinogen seem to be the rule in thisdisease, and we have also observed high levels ofserum globulin in a series of nine patients withacute rheumatism accompanied by sedimentationrates of more than 100 mm. in 1 hour (See TableI). However, the fact that high protein valuesaccompany high sedimentation rates in no way
proves a causal relationship.In order to determine whether one of these fac-
tors was actually the cause of the high sedimenta-tion rates in rheumatic fever, we attempted toreproduce the conditions necessary for rapid sedi-mentation by the modification of normal plasma(or serum) in various ways, including the addi-tion of protein fractions isolated from the bloodof normal individuals and patients with rheuma-tism. The sedimentation rates of washed humanerythrocytes resuspended in modified plasma were
then measured under standard conditions, in com-
parison with parallel measurements taken on redcells resuspended in the unmodified plasma ofnormal individuals and rheumatic patients.
The following technical precautions were observedthroughout:
1. All the cells and sera used in any one experimentwere of the same blood group.
2. Erythrocytes were washed three times with isotonic
NaCi, kept in the refrigerator, and used only if less than48 hours old.
3. Isotonicity was assured by dialyzing all proteinfractions overnight against 0.85 per cent NaCl.
4. Sodium citrate was used as anticoagulant.5. Long glass tubes (500 mm.) were used for sedi-
mentation, to minimize the effect of packing.Protein fractions were prepared as follows. Fibrino-
gen was precipitated from whole citrated plasma by theaddition of an equal volume of saturated NaCi. Thisprecipitate was dissolved in water and dialyzed, firstagainst distilled water to remove excess salt, then againstisotonic saline, and finally against isotonic saline underslight vacuum to reduce the volume to the desired level.The dialysis procedure was the same for all fractions.Plasma globulin was precipitated from the supernatant
fluid, after the removal of fibrinogen, by full saturationwith NaCl. The precipitate was dissolved in distilledwater and dialyzed.Serum globulin was precipitated from serum by satura-
tion with NaCl, dissolved in water and dialyzed. Inrapidly sedimenting blood this fraction included a smallamount of "residual" fibrinogen; i.e., a protein left insolution after clotting was complete, which could beprecipitated by one-half saturated NaCI.
Globulins were not subjected to further fractionation.The albumin fraction included all protein left in the
supernatant after full saturation with NaCl. Dialysiswas performed as usual.
Certain factors were found to play no signifi-cant part in the sedimentation mechanism. (1)Total seruim lipoids: Serum defatted by Hartley's(18) method produced a sedimentation rate equalto that of untreated serum, in the case of both aslowly and a rapidly sedimenting blood. Similarobservations have been recorded by Theorell (19)
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BLOOD SEDIMENTATION RATE IN RHEUMATIC CARDITIS
and by Ohlson and Rundquist (20). (2) Serumcomplement: Inactivation of serum by heating at560 for 30 minutes produced only slight changesin sedimentation rate.
Serum of rheumatic patient ........ .......... 80 mm.Inactivated serum of same patient ...... ...... 75 mm.
(3) Plasma crystalloids: Dialysis of whole plasmaor whole serum did not affect the sedimentationrate provided that isotonicity was restored andthat the concentration of total protein was notchanged.
Certain factors which have been shown byprevious workers to affect the sedimentation oferythrocytes were investigated in " reconstructed "bloods (plasma or serum plus washed cells), andwere found to operate in the usual way. Thesedimentation rate was slowed by increasing theratio of red cells to plasma, and vice versa. Re-duction of the total volume of normal plasmawithout increasing the salt concentration (bydialysis under negative pressure against isotonicsaline), also diminished the sedimentation rate.
Whole plasma .... ............................. 45 mm.Same, reduced to , of original volume, iso-
tonicity maintained ......... ............ 30 mm.
It was also found that dilution of plasma withisotonic salt (or Ringer's solution) resulted in amarked retardation of the sedimentation rate.Dilution of plasma to one-half or one-third of itsoriginal concentration resulted in a sedimentationrate approximately equal to that of the diluentalone. This is illustrated in Table II. The sedi-mentation rates are expressed as millimeters in 30minutes.The removal of fibrinogen from the plasma ob-
tained from normal or rheumatic patients slowedsedimentation considerably; nevertheless, sera
TABLE II
The effect on the sedimentation rate of diluting plasma withphysiological salt solutions
Albumin (i.e., serum from which the globulinhad been removed) inhibited sedimentation al-most completely.
Sample APlasma ................ ............. 135 mm.Serum....... 63 mm.Serum minus globulin ....................... 8 mm.
Sample BPlasma ...................... 100 mm.Plasma minus fibrinogen and globulin.9 mm.
It was not clear whether the disappearance ofthe sedimentation factor from serum on removalof globulin was due to the absence of globulinper se, or whether the inhibitory effect of dilutionwith saline was coming into play. This point wasinvestigated further by incorporating fibrinogenor globulin fractions, or both, into equivalentquanltities of normal plasma. As the final volumeof the modified plasma was the same as that ofthe original normal plasma, the protein concen-tration of the modified plasma was higher thanbefore. If the fractions used for modificationhad been inactive, their addition to normal plasmashould have inhibited sedimentation, owing to theincrease in the concentration of total protein.But the sedimentation rate of modified plasmawas in every case higher than that of normal 2plasma. The observed effects of adding or re-moving globulins must therefore be related to thepresence or absence of these particular proteins,and not merely to changes in the relative amountsof total protein and salt. Typical experimentsare presented in Table III.
2 The variability of the readings recorded for unmodi-fied normal plasma is due to variations in the density ofdifferent lots of cell suspensions used for the tests. Thesedimentation rates of all normal blood samples used, asdetermined by the Westergren technique, were less than20 mm. in 1 hour.
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ALVIN F. COBURN AND E. M. KAPP
TABLE III
The effect on the sedimentation rate of modifying normalplasma by the addition of various protein fractions
from normal and rheumatic blood
Experiment 1mm.
Normal plasma ................................... 55Normal plasma plus fibrinogen and globulin from an
equal volume of normal plasma .... ......... 95Experiment 2
Normal plasma ................................... 35Normal plasma plus fibrinogen from equal volume of
rheumatic plasma ........ .................. 82
Experiment 3Normal plasma ................................... 17Normal plasma plus fibrinogen from rheumatic
plasma ......... ........................... 43Normal plasma plus globulin from rheumatic plasma 30
Experiment 4Normal plasma .................................. 5Normal plasma plus fibrinogen from rheumatic
plasma.60Normal plasma plus globulin from rheumatic plasma 12Plasma from rheumatic patient ...... .............. 140
Fibrinogen was definitely more effective thanglobulin in every case studied; nevertheless theglobulin fractions showed considerable activity.However, the sedimentation rates of modifiednormal plasmas were in no case as high as thoseof the rheumatic plasmas from which the variousfractions had been prepared. This may be at-tributed to partial denaturation, especially offibrinogen, incident to precipitation, dialysis andother handling. In this connection, it may be ofinterest to note that the activity of serum sepa-
rated from clotted blood was slightly higher thanthat of serum obtained by defibrination.
Similar experiments have been reported byZ'arday and Farkas (21), who modified normalwhole blood by the addition of graded amounts ofnormal fibrinogen, globulin and albumin. Theyfound that the sedimentation rate was increasedin proportion to the amount of fibrinogen andglobulin added. Extra albumin, in contrast, re-
duced the rate. Our findings in acute rheumatismare entirely in accord with those 'of Z'arday andFarkas for normal blood. It is therefore con-
cluded that the interpretation of Fahraeus (14),that changes in sedimentation rate depend on
changes in the fibrinogen and globulin fractions ofplasma, is applicable to acute rheumatism.The mechanism whereby these proteins accel-
erate sedimentation is a matter of much contro-
versy, and need not concern us here. A fulldiscussion of the subject with complete bibliog-raphy is to be found in a recent monograph byReichel (22).
Immunological studies on fibrinogen and globulinfrom rheumatic patients
The experiments just described indicated quan-titative differences between the major proteinfractions of normal (slowly sedimenting) andrheumatic (rapidly sedimenting) bloods. Theydid not show whether there might be qualitativedifferences. In order to investigate this point, anexperiment was set up to detect immunologicalspecificity of fibrinogen and globulin in rheu-matic versus normal individuals, by means ofprecipitin tests with antisera to these proteins be-fore and after absorption with homologous andheterologous protein fractions.
Fibrinogen and globulin fractions were obtainedfrom the plasma of two classes of subjects: (1)Patients with acute rheumatism, with sedimenta-tion rates of more than 100 mm. in 1 hour; and(2) normal, healthy individuals with normal sedi-mentation rates. The protein solutions were alladjusted to contain equal concentrations of nitro-gen. A portion of each sample was used forimmunizing two rabbits, the remainder was keptsterile in the ice box. Strong precipitating anti-sera were obtained after four weeks of immuni-zation. These antisera were divided into threeparts. One part was stored untreated. Onepart was absorbed with its homologous antigen.The third part was absorbed with the correspond-ing protein fraction from the other class of sub-ject. Absorptions were performed at 370 C.After centrifugation, precipitin tests were set upwith the supernatant serum.
These tests showed no distinction betweennormal and rheumatic fibrinogen or betweennormal and rheumatic globulin. It was not possi-ble to absorb the antiserum to a fraction from arheumatic patient with its normal equivalent andobtain a supernatant fluid which would give aprecipitin reaction only with the homologousantigen. If absorption was complete, the serumcould no longer be precipitated by either type. Ifincomplete, the serum could be precipitated equallywell by protein from both normal and rheumaticindividuals. Sample protocols are presented in
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BLOOD SEDIMENTATION RATE IN RHEUMATIC CARDITIS
TABLE IV
Precipitin reactions between plasma protein fractions and their antisera, before and after absorption*
Rabbit antiserum to rheumatic globulin D, undiluted
* Four readings are given for each test: (1) after 20 minutes at room temperature; (2) after 2 hours' incubation at37.50 C.; (3) after 18 hours in the refrigerator; (4) after centrifugation.
Table IV. A qualitative change in the fibrinogenand globulin fractions of the blood in acute rheu-matism was not demonstrable by this method.The possibility of detecting such a change bymore refined procedures remains open.
DISCUSSION
Having explained the high sedimentation ratesof acute rheumatism as the result of increasedplasma fibrinogen and globulin, our next problemis to account for the change in the proteins. Atthis point direct evidence comes to an end, and wecan only look to the work of other investigatorsfor possible analogies.There is some evidence to indicate that fibrino-
gen and globulin are produced by the reticulo-endothelial system. For example, sharp, transientincreases in fibrinogen occur within two hours af-ter the injection of substances which are taken upby reticulo-endothelium, according to Held andBehr (23), whereas " blocking " of the reticulo-endothelial system by the previous injection ofcolloidal copper prevents this response. Fromthese and other experiments it seems that an in-crease of fibrinogen may be a direct response ofreticulo-endothelial cells to stimulation.
Increases in sedimentation rate during and fol-lowing hemolytic streptococcus respiratory infec-tions occur under three different clinical condi-tions. First, during the initial infection there may
be a mild rise in sedimentation rate which prob-ably represents the response of reticulo-endothe-lium to foreign substances in general. Second,during septic complications there is a further in-crease in sedimentation rate which may occur. inresponse to further invasion of the host by theinfectious agent. The third condition is fulfilledby sterile inflammatory processes such as acuterheumatism, the onset of which is accompaniedby a steep rise in the sedimentation rate curve toa high level. The intensity of this response sug-gests that a mechanism may be involved whichdiffers from that in the first two conditions.A number of observations have been made by
independent authors which may apply to the de-velopment of the high sedimentation rate in acuterheumatism. One of these is Berger's (24) find-ing, that the second of two equal injections offoreign protein is followed by a much larger in-crease in serum globulin than the first, when thesecond dose is given after the complete subsidenceof the globulin response to the first. The globulincurve following widely separated injections offoreign protein is similar to the sedimentation ratecurve of pharyngitis followed by acute rheuma-tism.
Another possible analogy which presents itselfis the development of high sedimentation rates bytuberculin-sensitive patients (15) or tuberculousrabbits (25) in response to injections of old tu-
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ALVIN F. COBURN AND E. M. KAPP
berculin. In these instances, as in rheumatism,the inflammatory process appears to be sterile andthe sedimentation rate rises rapidly to high levels.Normal controls show no rise in sedimentationrate when similarly injected. Roch (26) has re-cently described a similar response following re-peated injections of swine serum into rabbits. Inhis series, increases of sedimentation rate werelargely confined to skin-sensitive animals (Arthusphenomenon).The above findings all indicate that sharp in-
creases of sedimentation rate can be expected inresponse to repeated doses of foreign protein.The rheumatic subject necessarily receives a doseof foreign protein during acute pharyngitis. Asecond dose of the same foreign protein receivedat the end of Phase II would account for the ob-served increase in sedimentation rate at that time.Whether there is a second dose, and if so, the na-ture of the protein involved, remain to be estab-lished.
SUMMARY
In acute rheumatism, the sedimentation ratemay be considered as a measure of the extent ofinflammation.The increased sedimentation rate in acute rheu-
matism is caused by an increase in plasma fibrino-gen and globulin.An immunological test for a qualitative differ-
ence between the plasma protein fractions of nor-mal and rheumatic individuals gave negative re-sults.A possible type of mechanism is suggested to
account for the rise in sedimentation rate justbefore the onset of a rheumatic attack.
The authors are indebted to Dr. A. B. Gutman andEthel B. Gutman for a number of estimations of serumprotein.
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