OUR STUDIES on the seeming loss of

Transcription

1 Evaluatin f Bld Vlume Measurement Techniques By JOHN W. REMINGTON, PH.D., AND CARLETON H. BAKEB, PH.D. OUR STUDIES n the seeming lss f plasma frm the circulatin after transfusin and the mbilizatin f plasma ften seen after hemrrhage r drug infusins have been handicapped by a large variatin in measured respnses between animals. Cnversatins with ther wrkers n bld vlume regulatin have indicated that such variability is nt uncmmn. The necessity f using large grups f animals t test the effect f any experimental change makes fr tedius research. It als raises the questin as t the quantitative accuracy f the techniques used fr measuring bld vlume. We have, therefre, made several cnsecutive injectins f T 1824 and Cr 51 -tagged red cells in the same dgs t measure the ability f the tw techniques t reprduce the same vlume. This was then fllwed by a study f the ability f the tw t fllw knwn reductins in bld vlume prduced by stepwise hemrrhage. We have shwn 1 that after an injectin f tagged cells int dgs under mrphine-sdium pentbarbital anesthesia, the bld activity level falls t a fairly stable value in abut 10 minutes in splenectmized animals and in 20 t 30 minutes in intact dgs. Variatins f individual samples, when crrected fr any hematcrit change, abut the average plateau value have a standard deviatin f 3 per cent. Since the hematcrit technique shws a standard deviatin f 2 per cent, errrs attributable t an uneven distributin f tagged cells in the circulatin wuld appear t be small. Injected T 1824 des nt, f curse, reach a stable cncentratin after mixing is cmpleted, but cntinues t disappear thrugh the capillary membranes at what has been claimed t be an expnential rate. 2 ' 3 If the Frm the Department f Physilgy, Medical Cllege f Gergia, Augusta, Gergia. Supprted by Research Grant H-4573 frm the Natinal Heart Institute, U. S. Public Health Service, Bethesda, Maryland. Received fr publicatin July 5, rate f this lss can be clearly established, frm a semilgarithmie plt f cncentratin values ne culd extraplate back t the time f injectin and thus btain a reliable plasma vlume. Past studies in this labratry have frequently prduced dye-disappearance curves which shw transient increases in cncentratin lng after the'' mixing perid'' is claimed t be ver. 4 If such deviatins mark a prtracted mixing time, an extraplated slpe cnstructed n the basis f the available pints is rbbed f its theretical justificatin and becmes an empirical device nly. Dyecncentratin curves btained in the present study shw such deviatins, cming at varius times in the 70-minute sampling perid, t be the rule rather than the exceptin. Plasma vlume estimates based n the drawing f a best-fit slpe leave much t be desired as far as quantitative reprducibility is cncerned. Methds The dgs weighed frm 11 t 22 Kg., and were selected as the largest and apparently the healthiest f the animals available t us. They had been n an utside run with free access t an adequate water supply fr abut a week befre their use. In many eases, the spleens were then remved and the animals kept in individual cages fr ne t tw weeks mre. Tw types f experiments were dne. First, animals were given three r fur cnsecutive injectins f T 1824 and tagged red cells, separated by a sampling perid f 80 minutes. Three anesthetic regimens were used. Mst cmmnly we fllwed ur usual prcedure f giving 10 mg./kg. mrphine sulfate fllwed by an intravenus injectin f 15 t 20 mg./kg. sdium pentbarbital. Of 24 dgs s prepared, 11 were nrmal and 13 were chrnic splenectmized. A secnd grup f six dgs (fur intact, tw splenectmized) was given 200 mg./kg. sdium barbital intraperitneally (n mrphine). Tw full hurs were allwed t attain a stable anesthesia befre the first bld vlume determinatin was begun. All anesthetized dgs were placed n their backs n an animal bard and lightly tied fr the duratin f the experiment. A last grup f 10 splenectmized dgs was given n sedatin r anesthesia. They were gentle dgs, but nt table trained. Six were kept n their sides n the animal bard fr 60 Circulatin Research, Vlume IX, January 1961

2 BLOOD VOLUME MEASUREMENT TECHNIQUES 61 the duratin f the experiment (310 minutes), but they became restless befre the wrk was cmpleted. The last fur were allwed t remain n the flr except when samples were taken. We have nt been able t distinguish between the results btained with the six and the fur. In this first type f experiment, nly ne incisin was made ver the femral artery and vein f ne leg. Injectins were made int the vein, using syringes weighed befre and after delivery. At 10, 20, 30, 50, and 70 minutes after injectin, samples f 3.5 ml. were unifrmly taken frm the artery int 5 ml. syringes, fitted with 24 G needles, the barrels and needles f which had been wetted with istnic heparin slutin. Any samples shwing hemlysis r lipemia, which were rare, were discarded. The tagged red cells were made frm the animal's wn bld, as described previusly. 1 The T 1824 used was in a 0.15 per cent slutin, as freshly made up with distilled water frm an ampule f 0.5 per cent dye slutin (Lilly). Shrtly after remval, and after thrugh mixing, the hematerit value f each sample was determined in triplicate, using capillary tubes spun fr 10 minutes at 13,000 r.p.m. < The centrifuge and the specific technique have been described previusly. 1 The tubes were read with an Internatinal CR Header. N crrectin fr trapped plasma was cnsidered necessary. 1 A 1 ml. aliqut f whle bld was then pipetted int a test tube fr the determinatin f radiactivity, as described previusly. 1 The rest f the sample was then centrifuged, and the plasma pipetted ff. The plasma specific gravity was determined by the falling drp technique, and the T 1824 cncentratin was determined in undiluted plasma, using miereuvettes and a Beckman DU Spectrphtmeter. The read ptical density (O.D.) f each sample was crrected fr any change in plasma specific gravity away frm the value shwn by the blank sample taken just befre the injectin, subtracting frm each specific gravity befre making the crrectin by simple prprtin. The crrected O.D. values were then pltted n semilgarithmic paper, and the slpe f best fit drawn by the same investigatr fr all animals. The reference standard was made by adding 5 mierliters f the dye used fr injectin t 1 ml. plasma taken frm the blank sample. Plasma vlumes were calculated frm: (a) the O.D. value btained by extraplatin f the disappearance slpe t the time f injectin; (b) the O.D. value shwn by the 10-minute sample alne; and (c) the average O.D. value f the 10-, 20-, and 30-minute samples. At the end f the 70- minute perid, the ttal sample lss was divided int plasma and red cell lss accrding t the respective hematerit values. After subtracting Circulatin Research, Vlume IX, January 1961 the plasma lss frm the initially determined plasma vlume, the difference was in turn crrected fr any plasma specific gravity change (which was usually quite small in amunt) t give the expected new plasma vlume. The difference between the secnd plasma vlume determinatin and this expected value was then expressed in percentage f the latter. The expected cell vlume represented simply the value given by the initial determinatin less the cells remved in the samples. In the secnd type f experiment, 24 chrnically splenectmized dgs under sdium pentbarbital anesthesia had ne femral vein and artery expsed as abve and the ther femral artery expsed fr bleeding. The artic pressure was recrded by ptical manmeter frm the ascending arta by metal sund advanced thrugh a cartid artery, and the central venus pressure was recrded by saline manmeter thrugh a plastic tube advanced thrugh a jugular vein int the thracic cavity. Frm the recrded artic pressure pulses was calculated the cardiac index, 5 expressed in ml./min./m 2. The ttal peripheral resistance was calculated as the mean artic pressure in mm. Hg divided by the cardiac index. The zer level fr the venus manmeter was taken as that f the apex beat. Results and Discussin Cell Vlume Reprducibility The ability f successive injectins f tagged red cells t measure the expected cell vlume did nt appear t be influenced by the anesthesia (table 1) and the reprducibility appears quite acceptable. The difference in mixing time between intact and splenectmized dgs bserved befre 1 was seen here, but with five samples it was nt difficult t recgnize the plateau level. Fr all 34 dgs and the 84 attempted checks against the expected cell vlume, the standard deviatin was 4 per cent. This means that in any single animal, whether r nt it was splenectmized, a secnd determinatin had 19 chances in 20 f being within ± 8 per cent f the first. The size f this variability was nt set by any increase in the variatin f individual samples frm the mean with successive runs. This reprducibility is definitely better than that fund previusly fr 17 dgs in which ttal cell vlumes were calculated frm tw samples nly. 1 The tagged-cell methd measures directly the red cell distributin space. The ttal cell vlume is derived frm this by multiplying

3 62 REMINGTON, BAKER Reprducibility f Cell Vlume Determinatins Table 1 Sdium pentbarbital 11 intact dgs 1st 2nd 3rd l C.V.* (cc./kg.) Ht (%) C.V./H (cc./kg.) Expected C.V. (cc./kg.) } l (%) 1.6 ± ± Expected C.V./H (cc./kg.) J 2 (%) 1.4 ± ± splenectmized dgs Sdium barbital 6 dgs Un anesthetized 10 splenectmized dgs 1st 2nd 3rd 1st 2nd 3rd 4th 1st 2nd 3rd 4th ± ± ± ± ± ± ± ± it ± ± ± ± ± ± ± 2.2 *Cell vlume. thematcrit value. t± 1 standard errr. by the hematcrit value. We have used the arterial hematcrit fr this purpse. The riginal cell distributin space can be btained by dividing the cell vlume by the hematcrit (C.V./H) in table 1. If this space is taken t represent the ttal bld vlume, then the standard deviatin f the agreement between bserved and expected vlume is 3.0 per cent. The smaller deviatin is in large part attributable simply t the larger value fr bld vlume, s that a given discrepancy represents a smaller per cent change. Actually, because injected dye measures a larger bld vlume than d tagged cells, it is claimed that the hematcrit value fr the whle f the bld vlume (particularly in splenectmized dgs) is usually lwer than the arterial hematcrit. Crrecting the C.V./H values given in table 1 by 1/0.9 wuld make them all larger and the sample lss relatively less, but the effect n the bkkeeping wuld be f almst negligible prprtins. Cntrary t expectatin, 0-7 the calculated ttal circulatry hematcrit did nt remain cnstant thrugh the successive determinatins, but varied as the plasma vlume errr varied (see belw). Crrectin f the C.V./H values fr a changing ttal circulatry hematcrit wrsened the fit with the expected value. Plasma Vlume Reprducibility As in the past, we have nt btained with any cnsistency the rthdx type f dye-disappearance slpe. Fr example, the O.D. VA\- ues fr the five samples f the first plasma vlume determinatin fr 59 dgs under pentbarbital anesthesia were expressed in per cent f the 10-minute value. The btained curves grup themselves int five patterns, as shwn in figure 1. Only 17 cases shwed a decreasing dye cncentratin with each successive sample. At varius times after injectin, the thers shwed an increased dye cncentratin. It shuld be emphasized that these were all arterial samples, drawn withut stasis, and the O.D. value was crrected fr any change in specific gravity f the plasma. In many f these curves, the drawing f any disappearance slpe fr purpses f extraplatin is a highly subjective affair. In the reprdueibility studies, we have calculated plasma Circulatin Research, Vlume IX, January 1961

4 BLOOD VOLUME MEASUREMENT TECHNIQUES 63 UAntithfftitad A, l 10 dq» O SO SO Figure 1 Cntrl dye-disappearance curves as gruped int five categries. The pints represent the average values, as per cent f the value f the 10-minute sample. The transepts represent ± 1 standard errr. vlume by extraplatin f the best-fit slpe and, in a mre bjective manner, by use f the cncentratin at 10 minutes alne r f the average cncent: atin shwn by the 10-, 20-, and 30-minute samples. The results btained n 18 dgs are shwn in table 2. Successive dye injectins gave plasma vlumes greater than the expected values, the fit being wrse with the third injectin than with the secnd. If the trend is ignred, the standard deviatin f the 36 attempted checks against the expected values, as determined by the extraplated slpe prcedure, was 12.0 per cent r three times greater than that btained with the taggedcell injectins. Because the dye cncentratin at 10 minutes was usually abve the best-fit slpe, the average plasma vlume based n its value was almst the same as that by slpe extraplatin. As expected, the vlumes calculated frm the average cncentratin ver 30 minutes were greater. With the secnd plasma vlume determinatin, the relatins f the three calculated vlumes remained essentially unchanged, althugh the variability was greater with the tw bjective methds than with the slpe extraplatins. The 50- and 70-minute pints weighed heavily in the selectin f the best single slpe, and ur attempt t achieve a mre bjective measurement n the basis f certain samples nly was unrewarding. With the third determinatin, the disappearance slpe had bviusly steepened s that bth the Circulatin Research, Vlume IX, January 1961 too ZOO 300 TIME IN MINUTES Figure 2 Average dye-disappearance curves fr animals under mrphine-sdium pentbarbilal and sdium barbital anesthesia, and with n anesthesia. Fur cnsecutive dye injectins were made. Part (B) shws the ttal O.D. value fr each sample, as read against an undyed plasma blank. The pints pltted at the time f each injectin represent the expected O.D. n the basis f the amunt f dye injected and the amunt f dye and plasma lst in previus samples. The vertical line shws ± 1 standard errr frm this average expected value. Part (A) shics the O.D. values btained by subtracting the blank O.D. value (80-minute sample) frm each f the ttal 0-D. values f part (B) (slid pints) r by subtracting a decreasing blank O.D. value as taken frm extraplatin f the previus slpe (pen circles). The expected O.D. values ± 1 standard errr are given at the time f injectin. The numbers given under each curve represent the standard deviatin f the difference between actual O.D. value as btained by slpe extraplatin t zer time and the expected value as expressed in per cent f the expected. 10-minute O.D. value and the average O.D. value f the first three samples gave a greater plasma vlume than did the extraplated slpe. The steepening f the slpe represents a factr which was verlked at the time, althugh it shuld nt have been. 8 When, t determine the dilutin vlume f a secnd injectin f dye, the spectrphtmeter is nulled against a "blank" sample which cntains dye remaining frm the first injectin, the ma-

5 -4-64 REMINGTON, BAKER 5 S d 3? 00 ^ <-( a ^ - ^ i be u be I i -2^ i-h + 1 «O!> ' +i r-5 II O +t ci CM csi a n IS O «S d i I ^3 P 6 3 Ti g g 2 = S4 j, - (]) Pi chine is effectively subtracting a cnstant O.D. value frm each f the new series f samples. Yet, this previusly injected dye wuld cntinue t be lst, making the new disappearance rate appear mre rapid. Secndly, if the disappearance rate is expnential, then as the dye cncentratin becmes greater in the plasma, the abslute disappearance rate shuld als becme prprtinately greater. The relative rle f the tw factrs was cnsidered by a series f experiments shwn in figure 2. The spectrphtmeter was nulled against undyed plasma at the start, and then nt reset. Fr a first determinatin, samples were taken ver 70 minutes as befre, and a "blank" sample taken at 80 minutes. A new dye injectin was then made, and the ttal dye O.D. read n samples taken ver the secnd cllectin perid. Fur such injectins were made n each animal. The uncrrected O.D. values are shwn in figure 2B. The O.D. value f the "blank" was then subtracted frm each f the pstinjeetin samples t give the curve shwn in figure 2A. These subtractin curves shw the prgressive steepening nted in the previus experiments when the speetrphtmeter was nulled against the blank sample. Next, the best-fit slpe f the first sampling perid was prjected ver the time span f the next sampling perid, s that each pstinjeetin O.D. value had subtracted frm it an expected value taken frm this extraplated slpe. The difference is shwn by the pen circles f figure 2A. Unexpectedly, in abut half the cases, these pen circles shw variatin abut a cnstant value rather than a dwnward slpe. In these eases, then, the disappearance rate was seemingly nt changed bj r the injectin f mre dye, and the tw successive disappearance slpes were essentially parallel with a linear scale pltting, but nt with a lgarithmic scale pltting. In the rest f the cases, the pen circles did decline, but it still was nt always clear that the lgarithmic slpes were equal. These results culd mean that the disappearance slpe taken fr the first 70 minutes wuld nt cntinue thrugh the next 70 miu- Circulatin Research, Vlume IX. January 1961

6 BLOOD VOLUME MEASUREMENT TECHNIQUES 65 Figure 3 The individual dye-disappearance curves fr the fur successive determinatins fr the six dgs f figure 2 under sdium pentbarbital anesthesia. utes. It culd als mean that these slpes are nt necessarily expnential at all. By subtracting the dye remved in the samples taken and crrecting the dye cncentratin remaining in the dg fr any plasma specific gravity change, the expected ttal ptical density can be calculated. These expected values are given in figure 2 as pints pltted at the time f injectin, with a vertical line representing ± 1 standard errr. A priri, we wuld expect the lwer curves t give better quantitative agreement with these expected values than either f the upper curves. This was nt necessarily true. N matter hw calculated, with successive injectins there was still the strng tendency fr the dye cncentratins t be lwer than expected (r the plasma vlumes t be greater). Of the three grups f dgs, the unanesthetized animals came nearest t shwing parallel dyedisappearance slpes, yet the amunt f plasma vlume verestimatin was greatest. There is n apparent clue, then, in the disappearance slpe itself t the lack f plasma vlume reprducibility. The failure f reprducibility cannt be attributed, either, t a prgressive eliminatin f the srt f curve irregularities shwn in figure 1, which make the slpe determinatin difficult and might give false values fr the first plasma vlume determinatin. The average O.D. values fr the six dgs under sdium pentbarbital anesthesia given in figure 2A are shwn in figure 3. The steepening f the curves already referred t is quite bvius after the furth dye injectin. Increases in O.D. values late in the sampling perid are as cmmn with the later dye injectins as with the first ne. Circulatin Research, Vlume IX, January UJ <r w 100 <n UJ cc a. u 80 <r 60 u i 160 a! u -3 Systlic Cardiac index Heart Rate Central Venus Pressure 8.1 cc/kg 8.lcc/Kg cc/kg. Hemrrhage Figure 4 Hemdynamic studies n 24 dgs subjected t three bleedings. The transepts represent ± 1 S.E. f the difference frm the initial value. Vlume Estimatins after Hemrrhage Cntrl plasma (slpe extraplatin) and cell vlume determinatins were made in 24 splenectmized dgs. The animals were then bled rapidly frm the artery until the bld lss, when added t the previus sample lss, averaged 8.1 ml./kg. After 10 minutes were allwed fr circulatry cmpensatins fr the hemrrhage t be cmpleted, a secnd injectin f dye and tagged cells was made. The spectrphtmeter was nulled against a plas-

7 66 REMINGTON, BAKER IOO-I P.V. C.V. t i i Figure 5 Plasma vlume (P.V.) and cell vlume (C.V.) reductins in 24 dgs subjected t hemrrhage, expressed as per cent f the initial value. Crrectin f the P.V. fr change in plasma specific gravity gives the result pltted as the line (Pa); ivith ± 1 S.E. depicted by the transept. Crrectin by the amunt f hematcrit change gives the dtted line (Pn). The actual hematcrit and specific gravity changes are pltted belw. ma sample taken just befre the new injectin. After the sampling perid, a secnd hemrrhage f the same size was taken and a third injectin made. Finally, the dgs were bled by 15.8 ml./kg. and a furth injectin f dye and tagged cells made. As shwn in figure 4, the first tw bleedings caused little change in mean artic pressure but did prduce cardi-acceleratin, a rise in ttal resistance, and a decline in cardiac index and in central venus pressure. The last bleeding prduced a fall in artic pressure with less reductin in cardiac index, s that the ttal resistance decreased. These respnses t hemrrhage are nt unlike thse btained with smaller stepwise bleedings. 9 In figure 5 are shwn the plasma and cell 1st. bleeding 2nd. 3rd. Figure 6 Difference between the measured and expected plasma vlumes and cell vlumes in the 24 bled dgs, as expressed in per cent f the expected vlumes. The transept represents ± 1 S.E. The numbers given are the standard deviatin frm the expected vlume. vlume reductins accmpanying the sampling perids and the bleedings (lumped tgether), as expressed in per cent f the initial value as btained befre the hemrrhage. The bleedings were ften fllwed by rapid reductins in bth plasma specific gravity and in arterial hematcrit (fig. 5). Except after the first bleeding, the changes in the tw were f prprtinate degree, indicating an inward shift f ultrafiltrate but little mbilizatin f whle plasma. The expected plasma vlume shwn as the slid line in figure 5 was crrected upward by almst the same amunt if dne n the basis f the specific gravity change (PG), r f the hematcrit change (P H )- The measured vlumes were referred t these expected vlumes (P G ), expressing the difference again in terms f per cent f the expected. As shwn in figure 6, after the first bleeding the dye measured a vlume 4 per cent greater, n the average, than the expected but the size f the standard errr precluded any significance. After the secnd bleeding, the measured vlume was but 1.5 per cent greater. After the last bleeding, hwever, the measured vlume was significantly greater than expected and the standard deviatin very large. The tagged cells als verestimated the cell vlume by abut the same degree fr the first Circulatin Research, Vlume IX, January 1961

8 BLOOD VOLUME MEASUREMENT TECHNIQUES 67 tw bleedings but less (and withut statistical significance) after the final hemrrhage. The errr r red cell measurement was greater than with the reprducibility studies, but still was less than that f the plasma vlume determinatins. It has been claimed 10 that hemrrhage is accmpanied by a trapping f red cells in sme site nt available t injected tagged cells. Such a trapping wuld prduce the ppsite t what was seen in these experiments. Or it might be that the similar directinal changes f bth cell and plasma vlumes after hemrrhage wuld reflect a delayed mixing time. There is evidence that this is true as far as the cells are cncerned. In the prehemrrhage determinatin, the cell vlume was ± 0.4 per cent f the plateau level at 10 minutes. After the first bleeding, it was 97.4 ± 0.6 per cent at 10 minutes and ± 0.6 per cent at 20 minutes. After the last hemrrhage, the average vlume was 98.3 ± 0.7 per cent at 10 minutes, the same at 20 minutes, and finally ± 0.6 per cent at 30 minutes. But since the cell vlume used in the bkkeeping was based n the plateau level, whenever it was reached, ne culd hardly say that the delayed mixing was really determining the tendency fr the cells t verestimate the cell vlume. If the trend in cell vlume and in plasma vlume had a cmmn physilgical basis, then the data presented culd be interpreted t mean that the errr f reprducibility f the dye technique was actually better in these bleeding experiments than was true fr the reprducibility studies dne n unbled dgs. In bth types f experiments, we are quite uncertain why the dye technique has behaved s badly. There is n clear evidence that the failure f clser agreement with the expected is directly related t the anmalus dye-disappearance curves which we find. In final analysis, the dye cncentratin is nt as great 10 minutes after injectin as we expect it t be. These results are ppsite t thse f Cruickshank and Whitfield, 11 wh fund that succeeding dye injectins gave less steep slpes and better dye recvery. Circulatin Research, Vlume IX, January 1961 Ttal Circulatry Hematcrit Cnsiderable evidence has been presented that in splenectmized dgs the ttal circulatry hematcrit (TCH) bears the same relatinship t an arterial r venus hematcrit thrugh varius prcedures, such as bleeding and transfusins. 7 ' s The average rati fr a whle series f dgs shwed but minr changes in the abve experiments, but the value fr an individual animal shwed n necessary cnstancy. Hence, in the reprducibility studies, the standard deviatin f the change in TCH/H rati frm the initial value (84 eases) was 5.2 per cent, which meant that subsequent determinatins had ne chance in 20 f being greater than 10.5 per cent f the first. In the hemrrhage experiments, after the first bleeding the standard deviatin f the rati frm the initial was 7.8 per cent, after the secnd bleeding it was 10.6 per cent, and after the third it was 15.8 per cent. Since three variables are cncerned in this rati, it wuld be difficult t establish just which ne was mst respnsible fr the large variatin, but certainly the plasma vlume is mst suspect. At least a variability f this rder makes ne; hesitant abut making physilgical interpretatins based n an alleged cnstancy f the rati. Summary T assess the ability f an injectin f Cr 51 - tagged red cells and ne f T 1824 t measure a standard part f the ttal bld vlume, tw types f experiments have been dne. First, three r fur successive injectins were made, separated by the sampling perid f 80 minutes, in bth intact and splenectmized dgs under different anesthetic regimes. The tagged cells gave ttal cell vlumes shwing n trend away frm the expected value, based n the first determinatin and the cells lst in the sample taken, and the standard deviatin was but 3.0 per cent. There was n real difference in this errr depending upn the anesthesia. The T 1824 measured plasma vlume tended t becme prgressively greater than the expected value with each injectin. This trend culd nt be crrected ut n the basis f either a plasma specific gravity r a hemat-

9 68 REMINGTON, BAKER crit change. The standard deviatin was three times larger than fr the cell vlume. Part f this variability might be related t fluctuatins in dye cncentratin away frm the bestfit slpe, as were quite cmmn at varius times during the sampling perid. Yet, plasma vlumes based n the cncentratin shwn at any particular time (e.g., 10 minutes) gave slightly wrse agreement with the expected. The secnd experiment invlved measurements f cell and plasma vlume after three successive bleedings. Here bth vlumes were greater than the expected, with the standard deviatin f the cell vlume still less than that f the plasma vlume. Curiusly enugh, T 1824 culd fllw the changes in plasma vlume in these bleeding experiments a little better than it culd reprduce the same vlume in essentially nrmvlemic dgs. References 1. BAKER, C. H., AND BEMINGTON, J. "VV.: Ble f the spleen in determining ttal bdy hematcrit. Am. J. Physil. 198: 906, GIBSON, M. I., AND EVANS, W. A.: Clinical studies f the bld vlume: I. Clinical applicatin f a methd emplying the az dye '' Evans blue'' and the spectrphtmeter. J. Clin. Invest. 16: 301, GREGERSEN, J[. I., AXD STEWART, J. D.: Simultaneus determinatin f the plasma vlume with T-1824 and the "available fluid" vlume with sdium thicyanate. Am. J. Physil. 125: 142, PICKERING, B. W., AND DOW, P.: Arteri-venus distributin f brilliant vital red and T-1S24 injected int dgs. Am. J. Physil. 161: 221, REMINGTON, J. W.: Vlume quantitatin f the artic pressure pulse. Fed. Prc. 11: 750, GREGERSEN, M. I., C'IZEK, L. J., AND ALLEN, T. H.: Prprtin f "extra plasma" in the eviscerate dg. Am. J. Physil. 175: 224, HUGGINS, B. A., SMITH, E. L., AND DEAVERS, S.: Distributin f red cells and plasma in the dg. Am. J. Physil. 191: 163, GREGERSEN, JI. I., AND EAWSON, E. A.: Bld vlume. Physil. Eev. 39: 307, REMINGTON, J. W., HAMILTON, W. F., CADDELL, H. M., BOYD, G. H., AND HAMILTON, W. F., JR.: Sme circulatry respnses t hemrrhage in the dg. Am. J. Physil. 161: 106, DUNN,.T. B., JR., DEAVERS, S., HUGGINS, B. L., AND SMITH, E. L.: Effect f hemrrhage n cell and plsisma vlume f varius rgans f the dg. Am. J. Physil. 195: 69, CBUICKSHANK, E. W. H., AND WHITFIELD, I. C: Behavir f T-1824 (Evan's blue) in circulating bld and a mdified methd fr the estimatin f plasma vlume. J. Physil. 104: 52, Circulatin Research. Vlume IX, January 1961