NASA Technical Memorandum 86834 

NASA-TM-86834 19860003488 

Plasma Volume Methodology: 

Evans Blue, HemoglobinHematocrit, and Mass Density 

Transformations 

J.E. Greenleaf and H. Hinghofer-Szalkay 

November 1985 

NI\SI\ 

National Aeronautics and 

Space Admlntstratton 

OCT 3 1 198') 

L1BR;f/Y, N~.'of\ 

~'r\l\'1PTON 'lL-iGIl"11 \ 

111111111111111111111111111111111111111111111 

NF00075 
NASA Technical Memorandum 86834 

Plasma Volume Methodology: 

Evans Blue, HemoglobinHematocrit, and Mass Density 

Transformations 

J E Greenleaf, 

H Hlnghofer-Szalkay, Ames Research Center, Moffett Field, California 

November 1985 

NI\S/\ 

National Aeronautics and 

Space Administration 

Ames Research Center 

Moffett Field, California 94035 
SUMMARY 

Deta1led descriptions of methods for measuring absolute levels and changes in 

plasma volume are presented along with derivations of pertinent equations. Reduction in variability of the Evans blue dye dilution technique using chromatographlc 

column pur1fication suggests that the day-to-day variability in the plasma volume in 

humans 1S less than ±20 mI. Mass density determination using the mechanicalosc1llator technique provides a method for measuring vascular fluid shifts continuously for assess1ng the density of the filtrate, and for quantifying movements of 

prote1n across microvascular walls. Equations for the calculation of volume and 

density of shifted fluid are presented. 

INTRODUCTION 

Determination of plasma volume (PV) w1th Evans blue dye (T-1824) llnked to 

album1n and measured spectrophotometrically was f1rst reported by Gregersen 

(ref. 1-3). Th1S techn1que was developed pr1marily for the Armed Forces. Essent1ally the same techn1que, w1th some refinement on the separat10n and analys1s of 

the dye, is 1n use today. The accuracy and rel1ab1lity of this dye method depends 

upon the stab1lity of plasma album1n content over the perlod of measurement, as well 

as upon the k1netics of the bond between the dye and album1n molecule. Slmilar 

results are obtained when the Evans blue techn1que 1S compared with the radio10d1nated (1-131) labeled album1n technique (refs. 4-7). When the PV is determ1ned 

from analys1s of one 10-m1n post1njection dye sample, good agreement is obta1ned 

w1th the zero-t1me extrapolation techn1que, uS1ng multiple postinjection blood 

samples (ref. 6). Evans blue dye subjected to the Ames test for carc1nogen1c1ty 

(refs. 8 and 9) gave negat1ve results (ref. 10). 

W1th the advent of radioact1ve tracers, PV has usually been measured with one 

of the 10dine 1sotopes (1-125, 1-131). The problem then ar1ses of the appropr1ateness of uS1ng even tracer quantities of radioisotopes in humans in nonl1fethreaten1ng situations. It 1S possible that "the r1sks of low levels of rad1ation 

are much greater than llnear extrapolations would 1nd1cate" (ref. 11). 

For these reasons we w1sh to reemphasize the usefulness of Evans blue dye for 

the measurement of PV and to present a technique Slightly modified from that of 

Campbell, Frohman, and Reeve (ref. 7) for analyzing the dye concentration. 

A second problem 1S the measurement of changes 1n PV over minutes and hours. 

Repeated dye 1njections result in saturation of the albumin-binding sites, creat1ng 

such h1gh pre1nfus1on plasma concentrations that injection of additional dye cannot 
be measured accurately. As a result, the short-term (hourly) changes in plasma 

volume have been calculated from sequential measurements of hematocrit (Hct) and 

hemoglobin (Hb) concentrations (refs. 12-14) and the Hct alone (refs. 15-17). In 

addition to the errors incurred in the measurement of Hct and Hb, and the probable 

error in using antecubital v.nou. blood values as representative of total blood 

volume levels, the act of drawine blood samples through a catheter may cause a 

change in vascular hemodynamics and change the Hct and Hb concentrations. In spite 

of these probable sources of error, use of the Hb-hct and Hct equations for calculating ohanges in PV have proven useful and will continue to be used. Derivations 

of these equations are presented. 

Recently, the development of instruments (ref. 18) for very accurate measurement of blood and plasma density has made possible not only a fast and precise 

method for measuring ohanges In these volumes from discrete blood samples (refs. 19 

and 20), but it also provides for the first time a continuous measurement of changes 

in PV (refs. 21-23). This measurement technique is also described fully. 

EVANS BLUE DYE AND ISOTOPE LABELING 

Evans Blue Dye and 1-131 Labeled Albumin 

Gregersen and associates (ref. 2) were the first to use Evans blue dye (first 

obtained from Dr. H. M. Evans) to measure PV and, subsequently, they developed the 

method in detail (refs. 1 and 3). Measurement of dye diluted in untreated plasma 

results in rather large errors caused by lipemia or other Interfering substances 

which absorb at 615 nm. Allen and colleagues (refs. 24 and 25) were probably the 

first to use a detergent, which displaces the dye from albumin, and a cellulose pulp 

matrix to absorb the unbound dye. This basic technique was refined by Campbell 

et al. (ref. 26) and forms the basis for our technique, WhiCh is described here in 

detail. 

Reagents: 

1. Disodium hydrogen phosphate, Na2HP04 (anhydrous): 2~ solution 

(20 g/1000 ml). 

2. Teepol-phosphate [Teepol 610 ooncentrate, Particle Data Laboratories, Ltd., 

115 Hahn St., Elmhurst, IL 60126, (312) 832-5658]: Add 20 g Na2HP04 to 30 ml Teepol 

and make up to 1000 ml with distilled water. 

3. Potassium phosphate, monobaSIC, KH2P04 (anhydrous): B% solution 

(8 g/100 ml). 

4. 1:1 Acetone-water solution. 

5. Solka Floc BW-40 (60 ~) suspenSion of purified 

Corp., 650 Main St., Berlin, NH 03570, (603) 752-4600]: 

Solka Floc in 1000 ml 2% Na2HP04 (Reagent 1). 

2 

wood cellulose [James River 

Suspend approximately 10 g 
6. Evans blue dye (T-1824) standard [Harvey Laboratory and Pharmaceuticals, 

113 West Wyoming St., Philadelphia, PA 19140, (215) 451-1600]: Same lot number that 

1S used for injection. Refrigerate. (If in doubt about the lot number being the 

same, a quantity of dye will always be left over from the vial used for injection.) 

Preparat10n of columns: 

1. Prepare five columns for each subject: one for the standard, one for the 

blank uS1ng the "pre" sample, and three for the "post" samples. The columns can be 

fabricated by a glassblower; they are 25 cm long and have a burette stopcock on the 

lower end of a 13-cm glass tube with a 1-cm ins1de diameter; attached to the top of 

th1S tube 1S a larger glass tube 7 cm long with a 2.5-cm ins1de diameter. 

2. Insert approx1mately 100 mg (3 cm) of uncompressed glass wool 1nto each 

column, wash the tube with distilled water, and pack the glass wool firmly above the 

small constr1ct10n above the stopcock w1th a large stirring rod to a he1ght of about 

0.5 cm. 

3. With a pipette (upside down), pipette about 12 ml of the Solka Floc suspenSlon 1nto the column to a height of about 5 cm. (The p1petting should be carr led 

out qU1ckly after shak1ng the Solka Floc suspension, as 1t tends to settle very 

qU1ckly.) 

4. Wash each column using 

pack the column. (If poss1ble, 

that they w111 be packed well. 

reused.) 

a pipette w1th approx1mately 10 ml of 2% Na2HP04 to 

prepare the columns a day in advance of the test so 

When washed well with 2% Na2HP04' the columns can be 

Preparation of Evans blue for 1nject10n: 

1. After the dye has dra1ned to the bottom of the vial, w1pe the outs1de w1th 

alcohol and break open. Fill a ster11e, 10-ml syringe w1th the dye, using a ster1le 

1B-gauge needle. Remove the needle and expel all air from the barrel of the 

syr1nge. Push the level of the dye to the very tip of the syringe and attach a 

ster11e 20-gauge needle, bevel up. Replace cover over the needle, and label the 

syr1nge w1th the subject's name. W1pe away fingerpr1nts, and we1gh the syr1nge, 

needle, and cover on an analytical balance. Record th1S we1ght to four deC1mal 

places. 

2. Del1ver the loaded and we1ghed syr1nge to the person doing the 1nJection. 

Be sure the needle cover is returned w1th the empty syringe. 

3. Wipe f1ngerpr1nts from empty syr1nge and needle, and cover and rewe1gh the 

syringe apparatus. Subtract the empty we1ght (minus the weight of dye in needle) 

from the filled we1ght to determ1ne the exact weight of dye injected. 

3 
Preparation of the plasma samples: 

1. Centrifuge the heparinized blood from the "pre" injection and the "post" 

injection samples (3500 rpm for 10 min) and draw off and save the plasma in an 

appropriately labeled screw-cap vial. 

2. To 1 ml of the "pre" sample in a 50-ml Erlenmeyer flask, add 0.2 ml of the 

Evans blue standard solution (Reagent 6). Mix by swirling and let stand at least 

2 min. 

3. Set up the following in 50-ml Erlenmeyer flasks (one for each column): 

a. "Pre" injection plasma.--1 ml (BLANK) 

b. "Pre" injection plasma--1 ml + standard (STANDARD) 

c. "Post" injection plasma--1 ml (TEST) 

d. "Post" injection plasma--1 ml (TEST) 

e. "Post" injection plasma--1 ml (TEST) 

4. Add 15 ml of Teepol-phosphate to each flask, washing any plasma from the 

sides of the flask; swirl gently for about 15 sec and mix well. 

Extraction: 

1. Transfer the contents of each flask gently onto the Solka Floc in the 

column with a Pasteur pipette so that Its surface is not disturbed. 

2. RInse the flask with 5 ml of Teepol-phosphate and add this volume to the 

column. 

3. When the level of the solution has reached the Solka Floo, add at least 

10 ml of 2% Na2HP04 to the column. ThIS "wash" must be sufficient to remove all 

lnterfering substances, or else cloudiness will result in the final eluate. 

4. Allow the "wash" to pass through the column until the flUId is 0.5 cm above 

the Solka Floc. 

Elution: 

1. Elute Immediately. 

2. Pipette 0.5 ml of 8% KH2P04 into a 10-ml volumetric flask. This phosphate, 

with the Na2HP04 from the column, buffers the pH of the eluate to 7.0. 

3. Gently transfer approximately 5 ml of the acetone-water solution into the 

column and allow it to pass down until the blue front passes through the Solka Floc 

and nears the bottom. 

4 
4. Place the 10-ml volumetric flask with K2P04 under the column and allow the 

blue front to pass it. (Do not let the top of the column become dry; add more 

acetone-water if needed.) 

5. Allow the columns to stand 15 min before completing the elution. 

6. Add 4-5 ml of the acetone-water solut1on 1nto the column and cont1nue the 

elution unt11 the eluate fills the flask to the 10-ml mark; all the blue dye should 

have passed out of the plasma. 

7. Read the solutions in a spectrophotometer at 615 nm. 

Calculation: 

where 

v = 

D = 

St = 

v = 

T = 

1.03 = 

Plasma volume (ml) = 

volume (wt) of T-1824 1njected 

v x 0 x St x v 

T x 1.03 

d1lution of STANDARD (0.2 ml of 1:50; therefore d1lut1on = 1:250) 

absorbance of STANDARD 

volume of sample extracted (1 ml) 

absorbance of TEST sample m1nus absorbance of BLANK 

factor introduced to correct for slow dye uptake by the t1ssues 

Blood volume = PV x 100 100 - (0.87 Hct) 

(1) 

(2) 

where 0.87 1S a correction factor (about 4% of the Hct is trapped plasma; therefore, 

the true volume of packed cells 1S 0.96 Hct; whole-body Hct is 0.91 x true packed 

cell volume; hence, 0.96 x 0.91 = 0.87), and Hct is the raw Hct. 

Ames Test for Carc1nogen1c1ty of T-1824 

The Ames assay procedure (refs. 8,9) uS1ng Salmonella typh1murium (LT2) stra1ns 

TA1535, TA1537, TA1538, TA98, and TA100 is about 80% to 90% rel1able in detect1ng 

carc1nogens as mutagens. The test has about the same reliab1lity 1n 1dentifying 

chem1cals that are not carc1nogen1c. Therefore, neither a positive nor a negative 

response proves conclus1vely that a chem1cal 1S or 1S not carcinogenic to humans. 

The test consists of compar1ng the number of h1st1d1ne-1ndependent colonies on 

treated agar plates w1th the number found on control plates. Since all the plated 

Salmonella 1ndicator organ1sms undergo a few cell diV1s1ons in the presence of the 

test chem1cal, the assay procedure must be cons1dered sem1quant1tat1ve. The estab11shment of mutagen1c1ty (carcinogenlc1ty) rel1es on the demonstrat1on of a mutagen1c dose-response relationship. The test chem1cals are assayed at several dose 

levels w1th1n a nontox1c dose range. Toxic1ty is present 1f there 1S clearing of 

the background growth "lawn," a decrease In the number of "revertent" colonies below 

the spontaneous baCkground, or formation of pinpo1nt colonies of surv1v1ng cells. 

5 
The test chemicals are considered mutagenic in the Salmonella/microsome pre1ncubation assay if they elicit a reproducible dose-related increase in the number of 

histidine revertents per plate in one or more of the five test trains. The dye 

samples (General Diagnostics Lot 4018031 and Harvey Laboratories Lot 10810) were 

tested initially in strain TA9S up to 200 pI/plate, then in all five strains up to 

200 p/plate, and, finally, 1n all five strains up to a dose of 1000 pI/plate 

(approximately 4600 ~g of anhydrous dye). Each dose for human injection contains 

22.6 mg of dye that 1S diluted into 2.5 to 4.0 liters of plasma. Neither of the two 

Evans blue dye samples caused any tOX1C or mutagenic effects in any of the three 

assay tests. 

Errors in T-1824 Technique 

The repeatability of PV measurements with T-1824 depends upon the constancy of 

the plasma albumin content over the tracer equilibrium period, and from one measurement per10d to the next. Results from our stud1es (refs. 6 and 13) have indicated 

that: (a) Both T-1824 space and 1-131 space, determ1ned from a single sample 

obta1ned 10 min after InjectIon, were comparable with the zero-time extrapolation 

values determined from ser1al sampling. (b) Four consecutive inject10ns of T-1824 

on alternate days, resultIng In residual dye bUIldup to 87% of one dose, d1d not 

SIgnIficantly affect the accuracy of measurement of Evans blue space. (c) Four 

consecutive injections of 1-131 on alternate days resulting in reSIdual radiation 

buildup of 85% of one dose, d1d not slgnificantly affect the accuracy of measurement 

of 1-131 space, and (d) T-1824 and 1-131 did not Interfere with each other when the 

two tracers were used simultaneously, and their results were comparable (refs. 6 

and 13). We have studIed the var1abllity of the basal PV (Evans blue space) in 

normal, healthy humans at varIOUS inte~vals up to 28 days (Table 1). The variability (±SE) between subjects ranges from 16 to 112 ml, whereas the constancy of the 

PV over 28 days ranges between -31 and +28 mI. The mean (±SE) of the absolute 

dlfferences between the seven palred measurements lS 8 ±7 mI. These f1ndings emphaSlze the constancy of the day-to-day PV in men and women llvlng their normal lives 

(Table 1). 

There are two additional problems wlth the albumin-labeled tracer methods. One 

lnvolves the constancy of the albumin content over the per10d of measurement. The 

net exchange of album1n can vary from 4 to 7% within a 60-min period wlth subjects 

at rest or undergoing mild exerCIse (refs. 27), and there can be an addlt10nal Sh1ft 

of albumin (-4% to -8%) durlng 10 min of maximal exercise (refs. 15,27,28). A 

comparIson was made between the percent change ln PV, determined from control and 

postmaximal-exercise Evans blue spaces, and the change in PV from the Hb-Hct equatlon. The postexerc1se Evans blue value was the concentration of res1dual dye 

remaining after the single preexerclse injection. There was essentially no positive 

relationship between the PV determ1ned from the two methods, which suggests that 

analysis of residual dye 1S useless as an estimate of PV immediately after heavy 

exerClse (ref. 29) and after 8 hr of water immerSIon (ref. 30). The second problem 

is that the use of these tracer techn1ques is limited when one must make repeated 

measurements of PV over short time intervals. Since the minimum equilibratlon tlme 

6 
for these tracers is 10 min, PV determ1nations at shorter intervals are d1fficult to 

perform accurately. It is doubtful whether determinations made at consecutive 

10-m1n 1ntervals would be accurate because of the progressively increasing tracer 

concentrat1ons in the succeeding pre1njection basel1ne samples. Clearly, another 

analytical techn1que for est1mating changes 1n PV at shorter intervals is needed. 

HemoglobIn-HematocrIt and Hematocrit Transformat1ons 

It is interest1ng that the early methods for estimat1ng PV and total blood 

volumes 1nvolved the measurement of Hb concentrat1on before and after the 1nject1on 

of hemoblobin Solut1ons (ref. 31). The relat1ve constancy of the number of red 

blood cells and their content of Hb prov1des the baS1S for two related alternat1ve 

methods for calculat1ng changes 1n PV over short-term 1ntervals (hours). They 

1nvolve the measurement of the Hb concentrat1on and Hct (the ratio of the packed 

cell volume to the cell volume plus PV), and measurement of the Hct alone. 

The first equat10n for the calculat10n of changes in PV uS1ng the Hb and Hct 

was publ1shed by Elk1nton, Danowski, and Winkler (ref. 12): 

rewr1tten as 

PV 

a 

(1 - Hkt2) 

= x (1 Hkt 1

) 

(1 - Hct ) a x (1 - Hctb) 

Strauss et al. (49) put 1t 1n the form of percent change: 

(1 - Hct2) x x 100 (1 - Hct 1) 

rewr1tten as 

% 6PV = 100 

(1 - Hct

a ) x (1 Hctb) 

where a = after fluid Sh1ft and b = before fluid Sh1ft. 

(3) 

(4) 

(5) 

(6) 

If the right-hand side of equat10n (6) is subtracted from 100, the percent 

change In PV 1S obta1ned directly. The derivation of the equat10n 1S presented in 

Table 2. In this equat10n the change in PV 1S calculated from the Hct, assum1ng the 

red cell volume and the Fcell rat10 (total body Hct/large vessel Hct) rema1n 

7 
constant and the total blood volume is equal to the sum of the cell volume and the 

PV. Use of the Hb concentration corrects for changes in red cell water content, 

i.e., the mean corpuscular volume. 

If the red cell volume and the Fcell 

Hct are a measure of the changes in PV. An 

in PV from Hct values alone is given by "an 

a proportionality factor [100/(100 - Hctb)] 

before (b) and after (a) ratios: 

% PV 100 

= x 100 - Hctb 

ratio remain constant, changes in the 

equation for calculating percent change 

Beaumont et ale (ref. 16). It involves 

and tue percent difference between the 

100(Hctb - Hct

a ) 

Hct

a 

(8) 

There have been numerous reports in which percent changes 1n Hct, calculated 

with only the percent difference factor, were equated with percent changes 1n PV 

(refs. 32-38). The results 1ndicated that the percent changes 1n Hct were always 

smaller than the percent changes 1n PV. Some explanat10ns for this d1screpancy 

between the two calculated values were a different1al rate of change between them 

(ref. 3), or some unexpla1ned problem with the slte of blood sampl1ng (ref. 35). 

Another explanation 1S that percent changes 1n plasma are calculated on the baS1S of 

the PV alone, wh1le percent changes 1n Hct are calculated on the baS1S of total 

blood volume, not just the plasma port1on (ref. 11). Use of the proport10nal1ty 

factor 1n equat10n (1) (Table 2) converts (l.e., reduces) the base from total blood 

volume to the PV, so now all compar1sons are made on the bas1s of the PV. ThUS, any 

variable measured on the baS1S of total blood volume (e.g., Hb) can be converted to 

a PV basis w1th the Hct proportionality factor, prov1ded the the Fcell rat10 1S 

known. 

If PV is measured and red cell volume 1S assumed to be constant, a posttreatment PV can be calculated (ref. 11), PYa = BVa - CVb, where BV 1S the blood 

volume, and CV 1S the red cell volume, and 

PV = 

a 

-2 [PVb/1 - (Hctb x 10 )] - PVb 

Hct x 10-2 

a 

10- 2 ) - PVb - (Hctb x 

(9) 

Also, 1f the percent change in PV 1S calculated in the convent1onal manner, 

i.e., % ~PV = [(PVb - PVa)/PVb] x 100, the percent change in any plasma const1tuent 

concentration (e.g., protein) must be calculated w1th the "after" value 1n the 

denom1nator (ref. 11): 

( 10) 

The correlation coefficient between measured PV and PV calculated from the Hct 

changes with data from five stud1es from the literature 1S better than 0.95 

(ref. 17). Finally, the percent change in the total content (Co) of a plasma constituent can also be calculated from the before-and-after Hct values and the 

8 
concentrat1ons (Cn) of the constituent being measured (ref. 28). The compos1te 

equation is as follows (ref. 29): 

% ~Co = ( 11) 

The major advantage of the Hct transformation is that it requires determ1nat1on 

of only the Hct. The deficIencies are that 1t assumes a constant red blood cell 

volume or mean corpuscular volume (MCV) and that the venous Hct 1S representative of 

the total body Hct. A correction factor of 0.91 is usually appl1ed in resting 

subjects to correct venous Hct to total body Hct (refs. 3 and 13), but changes 1n 

th1S factor would introduce errors 1n the calculated volumes (ref. 39). One way to 

prov1de some correction for potent1al changes in the mean corpuscular volume 1S to 

comb1ne measurements of Hct w1th those of red cell hemoglob1n. The Hb/Hct 1S a 

measure of the mean corpuscular hemoglobin concentration (MCHbCn) (ref. 40) and can 

be used to est1mate changes 1n the mean corpuscular volume (Hct/red blood cell (RBC) 

concentrat1on) when the RBC concentrat1on 1S not available (ref. 41). A stable 

MCHbCn would also 1nd1cate an unchanged MCV because a change 1n cell volume, result1ng from a shift of fluid 1nto the RBC, would change the RBC mass; the Hb concentrat10n would rema1n unchanged, wh1le the Hct and MCHbCn would change. 

Compar1son of Hct and Hb-Hct Methods for Calculat1ng Percent Change 1n PV 

There 1S not total agreement among stud1es for the calculated changes 1n PV 

w1th the Hct versus the Hb-Hct methods under some exerC1se and environmental cond1-

t1ons. On the bas1s of results obta1ned from stud1es on men undergoing exerC1se 

dehydrat1on, thermal dehydration, and submax1mal and max1mal exerC1se 1n a thermoneutral env1ronment, Cost111 et al. (refs. 42 and 43) concluded that the changes 1n 

PV calculated with the Hct method w1II be 1n error unless correct1ons are made for 

changes 1n the mean corpuscular volume. The1r data suggest that the MCV 1S reduced 

under these conditions because of 1ncreases 1n plasma osmolality. On the other 

hand, Van Beaumont and Rochelle (ref. 44) found no change 1n MCV with 1ncreases 1n 

plasma osmolallty of 12%, lnduced by maxlmal arm exerClse. It seems clear that 

short-term 1ntenS1ve stress has essent1ally no effect on MCV as measured by the 

regress10n of percent change In PV calculated with the Hct or the Hb-Hct. It 1S 

poss1ble that 1ncreased osmot1c concentrat1ons decrease the MCV 1n V1VO, but th1S 

hypothes1s has not been conf1rmed (ref. 45). Perhaps there 1S an upper homeostat1c 

Ilm1t, 1n V1VO, for 1ncreased osmolal1ty beyond WhlCh the MCV is unchanged. But a 

11near relationsh1p is unl1kely, because the integr1ty of the RBC volume would 

presumably be protected w1thln some well-def1ned "normal" range of plasma osmot1c 

var1ab1lity. The positive SIde of this normal range appears to be 0 to 13 mosmol/kg 

(ref. 13), and the Hct equation could be used to estimate changes in PV. W1th1n 

th1S osmot1c range fall values from studies 1nvolv1ng maximal exercise short-term 

(max1mal) +Gz acceleration, 10° tllting, SItting 1n a hot env1ronment, and 

submaxlmal exerC1se below 50% ~02 max for less than 60 mln. The probable effect of 

longer-durat1on stress (greater than 1 hr) on changes 1n MCV suggest factors other 

9 
than just an increase in plasma osmolality are involved, because osmotic equillbrium 

between plasma and RBe probably occurs within minutes, not hours. 

While the Hct and Hb-Hct transformations provide a method for estimating 

changes in PV at much shorter intervals than with Evans blue dye or lodine isotope 

dllution, they cannot be used to measure changes in PV continuously. Measurement of 

changes in blood density on-line can be ~sed to estimate changes in plasma or blood 

volumes continuously. This method wlil be discu&~ed in the followlng sectlon. 

BLOOD DENSITOMETRt 

Densitometry on Biologic Fluids 

Blood denslty (BO) measurements were first performed in 1881 with the fall Ingdrop method (ref. 46). In the late 1940s, slmilar technlques were used to estimate 

Hct, Hb, and plasma proteln concentrations (refs. 41 and 48). But because all early 

density technlques (refractometry, faillng-drop method) were elther Impreclse or 

laborious, densltometry had been abandoned for use in physiological research. For 

physical and industrial research purposes, the reliable mass and volume measurements, often hard to perform with the required accuracy, remained the method of 

chOlce for determlnatlons of a sample's mass density, l.e., mass per unlt volume. 

In 1966 a new method was introduced--the mechanical osclilator technique 

(MOT)--which was first used for high-precision measurements on homogenous flulds and 

gases in conjunctlon wlth X-ray refractlon analysis of molecular structure 

(ref. 18). Shortly thereafter, the MOT was used to measure the denslty of beverages, gasoline, tritiated water, inorganiC and organlc solutions, etc. Recently, 

ItS use for contlnuous measurement on blood (refs. 21-23,49-51) and lymph (ref. 52) 

was introduced. 

The MOT combines the advantages of hlgh accuracy (up to 10-6 g/ml); small 

volumes (~50 ~l); appllcabillty for contlnuous recordings; short measurlng tlme 

(~1 s per measuring cycle in the contlnuous mode, dependlng upon the dimenSlons of 

the osc1llator used); and independence from an external acceleratlon fleld, which 

allows its use in welghtless condlt1ons. With the Introduction of th1S method In 

biology, physiology, and clin1cal med1clne, h1gh-prec1sion denslty measurements of 

blood and its constItuents have rega1ned their usefulness for biomedical research. 

How the Mechanlcal Osc1llator Techn1que Works 

Mass density determinations with the denslty-measuring apparatus (OMA) employ 

the MOT, which 1S based on the mass-sprlng principle: the actual resonant frequency 

(f) of an oscillating U-shaped glass tube, comparable to a tunlng fork In shape and 

phYSIcal features, is determined accordlng to 

f = 1/2 ~/c/(M + O·V) o 

10 

(12) 
where c lS the constant of elasticity for the glass tube, Mo is the inertial mass 

of the empty tube (the oscillator), D is the density (mass per un1t volume), and 

V the volume of the sample contained in the tube. 

The tube lS brought to bend lng-type osclilatlons perpendicular to the plane of 

the U by a variable electromagnetic sinuso1dal field, and the movements of ltS tlP 

are sensed by an optical system. The resonant frequency is tracked by a feedback 

loop. The osclilation duration is determined precisely for each measurlng perlod by 

sensing and averaging several hundred slngle cycles on-line. The length of the 

measurlng period can be preset (e.g., =1, 2, 4, or 8 sec ln the case of low-volume 

DMAs). The MOT lS based on high-resolution oscillation time determlnatlons. 

Before startlng a set of density recordings, the system is callbrated wlth two 

medla of known denslty, usually alr and water. Then, density values are computed by 

the bUllt-ln microprocessor from the average oscillation time. The denslty slgnal 

can be dlrectly fed to a dlgltal/analog output for continuous recordlngs. Thus, 

hlgh-preclsion density measurements can be made on small volumes of blOloglC flulds 

wlthout mass or volume measurements. 

The vlbratlon of the DMA's glass tube creates centrifugal forces, the resultant 

of WhlCh 1S directed toward the tlP of the oscillator. Since thlS force lS «1 gram 

1n the normal operating range (ref. 53), no lnterference in terms of blood sedimentatlon occurs during BD measurements, particularly in the cont1nuous mode. 

Deposltlon of plasma protelns and corpuscular elements (e.g., thrombocyte 

aggregates) can be prevented by coatlng the glass surface wlth slilcon prlor to 

measurement uSlng SurfasllTM (Plerce Co.). No difference between contlnuously 

determlned BD and values from synchronously obtalned blood samples could be observed 

when coated osc1llators were used, especially for operation exceedlng several hours 

(ref. 21). 

Slgnlflcance of the Mass Denslty of B10log1C Fluids 

The denslty of lsotonlC flulds at a given temperature is a llnear function of 

the fluld's total proteln concentratlon (refs. 54 and 55); for blood thlS lS 

approXlmately 

BPC = PPC(1 - HctR - HctW) + MCHbCn . HctR + MLPC . HctW ( 13) 

where BPC lS the blood proteln concentratlon, PPC is the plasma proteln concentratIon, MCHbCn lS the mean corpuscular hemoglobln concentrat10n, MLPC lS the mean 

leukocyte proteln concentration, HctR lS the red cell hematocrlt, and HctW 1S the 

whlte cell hematocrit (given as volume fract10n). The contrlbutlon of blood platelets 1S lncorporated lnto the plasma protein concentratlon after mlld centr1fugatlon 

(platelet-rlch plasma), and into the mean leukocyte proteln concentratlon after 

lntense centrifugation of blood. If hemolysls occurs, the influence of additlonal 

Hb on the plasma density can be compensated for by colorimetrlc means (ref. 56) to 

determme the "true" plasma denslty. 

11 
The plasma protein and mean corpuscular hemoglobin concentrations are regulated 

within narrow limits under normal physiological conditions whereas Hct may vary more 

wIdely; Hctw is usually low «0.01). Since the mean corpuscular Hb concentration IS 

four to five times greater (300-400 gram/liter) than the plasma protein concentration (60-80 gil), blood protein and Hct are closely correlated. Consequently, the 

BD WhICh is a function of the blood proteIn concentration, is correlated lInearly 

with Hct (refs. 19,20,54). However, PD--~t a given temperature and proteIn concentration--is influenced by plasma lipid, glucose, and urea concentratIon (ref. 55). 

Further, the leukocyte density becomes more Important in cases of leukocytosis. 

These confoundIng factors may diminIsh the accuracy ~: densItometry for the determInation of protein concentratIon and Hct, although It remains effectIve for rough 

estimatIons, particularly on a screenIng basIs. 

In a single experIment, In which the amount of these potentially confounding 

factors can be assumed to remaIn constant, densIty alteratIons are a precise measure 

of plasma proteIn and Hb concentratIon shifts. Moreover, the densIty of fluId 

shifted between the extravascular and Intravascular compartments can be computed, 

SInce this "fIltrate" densIty differs from both blood and plasma densItIes (refs. 21 

and 39). ExperIments WhICh combine this technique WIth the advantage of contInuous 

BD measurements reveal new Insight not only Into the amount but also Into the timecourse of rapid mIcrovascular fluId exchange (refs. 21,23,50,51). Dur1ng contInuous 

densItometry, proper compensatIon of possIble thermal deviatIons becomes most Important. Recent studies (refs. 19 and 57) have provided data for correct1ons of temperature-induced errors In on-line density recordIngs. 

DensIty Shifts WIth Changes In Posture 

Blood volume regulat10n In humans IS serIously challenged by assuming an 

uprIght posture. QUIet standIng leads to rapid and persistent hemoconcentration, 

whIch may result in PV losses in excess of 20%. Calculation of the densIty of flUId 

leavIng or enterIng the cIrculatory system, under IsotonIC conditIons, allows for 

the assessment of the amount of proteIn WhICh moves across the cap1llary walls 

durIng orthostat1cally Induced capIllary filtratIon. 

The mass densIty of red cells does not change WIth alterations of posture 1n 

euhydrated man (ref. 58). ThIS observation helps to valIdate the calculatIon of the 

density and volume of flUId shIfted between 1ntra- and extravascular compartments, 

USIng formulae WhICh assume no net fluid transfer between the corpuscular and plasma 

compartments. For both Inward fIltratIon (from assumpt10n of the supIne poslt1on 

after the upright) and outward movement of flUId (uprIght POSItIon after supIne), 

essentially the same fluid denslty (1008 gram/liter, 37°C) IS found wlth an 

unchanged Fcell ratIO. This result lndicates a proteln concentration of ~30 gil 

In the shifted fluid, or approxImately 40% of that in the blood plasma (39). 

12 
Continuous Blood Density Measurements 

The MOT has been employed for continuous measurements on arterial blood in 

animals (refs. 21,23,50) and during blood dialysis in patients (refs. 23 and 49). 

Recently, coronary sinus blood in dogs was used for on-line densit~ recordings 

(ref. 51). The accuracy of the density data was better than 1·10- g/ml in these 

stud1es. 

Changes in the plasma protein concentration after blood volume alteratIons 

provide an accurate measure of volume replacement only if the net protein concentration of the shifted flu1d is known. Results from different hemorrhage studies are 

contradictory and demonstrate that filtrate protein content ranges from zero to 

about half of the plasma concentration after hemorrhage. If a slgn1ficant amount of 

prote1n moves with the filtrate, then the degree of fluid replacement 1S underestimated from calculations which are based on the assumption that the observed 

plasma prote1n concentration changes are due to an admixture of prote1n-free 

fluid. Continuous monitoring of the mass density of flow1ng blood, combined w1th 

plasma dens1ty and Hct determinat1ons, can help to elucidate the volume and prote1n 

concentration of even small flu1d shifts. Results from a recent study on the 

effects of moderate hemorrhage (ref. 21) suggested that e1ther the 1nward-shlfted 

flu1d had a h1gher density than normal ultrafiltrate, and/or there was a rise of the 

rat10 of whole-body to large-vessel Hct (Fcell ratio). 

The investigation of rapid compensatory mechanisms after blood loss 1S complicated by the poss1bility of simultaneous shifts in the Fcell rat10. If It IS 

altered, the observed changes in measured Hct are due only in part to transcapillary 

fluid flux, and volume shift results from the Hct data may be 1n error. 

If the Fcell ratio is constant, as dur1ng pass1ve body t1ltlng, the denSIty 

of transcap1llary exchanged fluid can be calculated from blood denSIty (BD) and 

plasma dens1ty (PO) changes (refs. 21 and 39) uS1ng the formulae: 

Hct'(1 - HctO) 

FD = PdO - Hct

o _ Hct' (PDO - PO') (14) 

or 

FD = BD - Hct' (BD B ') o Hcto - Hct' 0 - 0 (15) 

where PDO' HctO' and BOO refer to the state wIth the larger blood volume (after 

volume repletion, or before outward shift), whereas PO', Hct', and BD' refer to 

the hemoconcentrated condit1on. Hct 1S corrected for whole body hematocrIt. 

Provided there are no measur1ng errors and the red cell volume rema1ned constant, 

the flu1d density (FD) from both equations should be identical. 

Whereas plasma density and Hct cannot be measured continuously, the mechanical 

oscillator techn1que provides the advantage of precise on-line measurement of blood 

density changes. As an example, figure 1 shows the first blood density curve 

13 
measured in a human which was obtained from venous (cubital) blood when the subject 

changed from the sitting to the standing and the supine positions. There are transient deviations from a simple exponential time-course curve immediately after the 

postural changes which may be caused by baroreflex-driven dynamic resettings of the 

peripheral reSistance, thus influencing the capillary filtration pressure on a 

minute-by-m1nute basls, 1n addltlpn to the normal hydrostatic effects on mIcrovascular fluid exchange. 

From the magnitude of the density alterations, the volume of the exchanged 

fluid (FV) can be calculated from 

BOO - BO' 

FV ; ~~---",.,,- BO' - FO (fraction of BVO) (16) 

Thus, WIth use of blood densitometry, not only the magnitude, but also the 

continuous time-course of flUId ShIfts can be detected. Use of this technIque 

should result In sIgnificant advances in our knowledge of fluId-electrolyte-proteln 

physiology. 

14 
REFERENCES 

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6. Young, H. L.; Juhos, L.; Castle, B. L.; Yusken, J.; and Greenleaf, J. E.: Body 

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Tech. Rept. R-406, WashIngton, DC, 1913, 19 pp. 

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9. Ames, B. N.; Lee, F. D.; and Durston, W. E.: An Improved Bacterlal Test System 

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Acad. Sci., vol. 10, 1913, pp. 182-186. 

10. Juhos, L. T.; Mortelmans, K. E.; and Shepherd, G. F.: In Vltro Mlcroblologlcal 

MutagenIcIty Assays of Evans Blue. Stanford Res. Inst. Int. Rept., LSU-1449, 

1980, 12 pp. 

11. Marx, J. L.: Low-Level Radlation: Just How Bad Is It? SClence, vol. 204, 

1919, pp. 160-164. 

15 
12. Elkinton, J. R.; Danowski, T. S.; and Winkler, A. W.: Hemodynamic Changes ln 

Salt Depletion and in Dehydratlon. J. Clin. Invest., vol. 25, 1946, pp. 

120-169. 

13. Greenleaf, J. E.; Convertino, V. A.; and Hangseth, G. R.: Plasma Volume During 

Stress 1n Han: Osmolality and Red Cell Volume. J. Appl. Physiol., vol. 47, 

1979, pp. 1031-1038. 

14. Strauss, H. B.; Davis, R. K.; Rosenbaum, J. D.; and R08smeisl, E. C.: "Water 

Diuresis" Produced During Recumbency by the Int~Qvenous Infuslon of Isotonlc 

Sal1ne Solution. J. Clin. Invest., vol. 30, 1951, pp. 862-868. 

15. Greenleaf, J. E.; Bernauer, E. M.; Adams, W. C.; and Juhos, L.: Fluidelectrolyte shift and V02 max ln Han at Slmulated Alt1tude (2,287 m). J. Appl. 

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16. 

17. 

18. 

19. 

20. 

21. 

Van Beaumont, W.: Evaluation of Hemoconcentration From Hematocrlt 

Measurements. J. Appl. Physiol., vol. 32, 1972, pp. 712-713. 

Van Beaumont, W.; Greenleaf, J. E.; and Juhos, L.: Disproportlonal Changes ln 

Hematocrit, Plasma Volume, and Protelns During Exercise and Bed Rest. J. Appl. 

Physiol., vol. 33, 1972, pp. 55-61. 

Kratky, 0.; Leopold, H.; and Stablnger, H.: Dichtemessung an Flusslgkelten und 

Gassen auf 10-6 g/cm3 bei 0.6 cm3 Probenvolumen. Zeit. Angew. Physlk, vol. 27, 

1966, pp. 273-277. 

Hinghofer-Szalkay, H.; and Holzer, H.: The Calculation of Hemoglobln 

Concentration from Blood and Plasma Densltles, Measured by the Mechanlcal 

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pp. 613-618. 

Hinghofer-Szalkay, T.; Kenner, T.; Leopold, H.; and Holzer, H.: Ole Anwendung 

der Blegeschw1ngermethode zur Messung von Blutdichte und Hamatokr1t. KIln. 

Wochenschr., vol. 57, 1979, pp. 1163-1167. 

Hinghofer-Szalkay, H.: Contlnuous Blood Densltometry: Flu1d Shlfts After 

Graded Hemorrhage in Animals. Am. J. Physiol.: Heart eirc. Physlol., 1985. 

In press. 

22. Kenner, T.; Hinghofer-Szalkay, H.; Leopold, H.; and Pogglitsch, H.: Verhalten 

der Blutdichte ln Relatlon zum Blutdruck im Tierversuch und bel Hamodialyse von 

Patienten. Zelt. Kardiologle, vol. 66, 1977, pp. 399-401. 

23. Kenner, T.; Leopold, H.; and Hinghofer-Szalkay, H.: The Contlnuous HlghPreclsion Measurement of the Denslty of Flowing Blood. Pfluegers Arch., vol. 

370, 1977, pp. 25-29. 

16 
24. Allen, T. 

Lipemia. 

J.: Extraction of T-1824 in the Presence of Gross Hemolysis and 

Proc. Soc. Exp. Biol. Med., vol. 16, 1951, pp. 145-141. 

25. Allen, T. H.; and Orahovats, P. D.: Cellophane Measurement of T-1824. Am. J. 

PhYSI01., vol. 154, 1948, pp. 21-37. 

26. Campbell, T. J.; Frohman, B.; and E. B. Reeve: A SImple, RapId, and Accurate 

Method of Extracting T-1824 from Plasma, Adapted to the Routine Measurement of 

Blood Volume. J. Lab. Clin. Med., vol. 52, 1958, pp. 768-777. 

27. Greenleaf, J. E., Convertino, V. A.; 

C.; Vignau, S. R.; and Brock, P. J.: 

and Thermoregulat1on During Exercise 

pp. 1026-1032. 

Stremel, R. W.; Bernauer, E. M.; Adams, W. 

Plasma [Na+], [Ca2+] and Volume Sh1fts 

in Man. J. Appl. Physiol., vol. 43, 1977, 

28. Van Beaumont, W.; Strand, J. C.; Petrofsky, J. S.; Hipskind, S. G.; and 

Greenleaf, J. E.: Changes in Total Plasma Content of Electrolytes and Prote1ns 

w1th MaxImal Exerc1se. J. Appl. Physiol., vol. 34, 1973, pp. 102-106. 

29. Greenleaf, J. E.; Van Beaumont, W.; Brock, P. J.; Morse, J. T.; and Mangseth, 

G. R.: Plasma Volume and Electrolyte Sh1fts With Heavy Exerc1se 1n Sltting and 

Supine POS1tions. Am. J. Physiol., vol. 236 (Regulatory IntegratIve Compo 

PhYS10l., vol. 5), 1979, pp. R206-R214. 

30. Greenleaf, J. E.; Shvartz, E.; and Keil, L. C.: Hemod1lutlon, VasopreSSin 

Suppression, and D1uresis Dur1ng Water Immers10n in Man. AVlat. Space Env1ron. 

Med., vol. 52, 1981, pp. 329-336. 

31. Barratt, J. 0. W.; and Yorke, W.: A Method of Estimat1ng the Total Volume of 

Blood Contained 1n the LIv1ng Body. Proc. Roy. Soc. London, vol. 81, 1909, 

pp. 381-394. 

32. Bullard, R. W.; D1ll, D. B.; and Yousef, M. K.: Responses of the Burro to 

Desert Heat Stress. J. Appl. PhYS10l., vol. 29, 1970, pp. 159-161. 

33. Joye, H.; and Poortmans, J. R.: HematocrIt and Serum Prote1ns Dur1ng Arm ExerCIse. Med. Sci. Sports, vol. 2, 1970, pp. 187-190. 

34. Poortmans, J. R.: Serum Prote1n Determ1nat1on During Short Exhaustive Phys1cal 

Act1Vity. J. Appl. PhYS10l., vol. 30, 1970, pp. 190-192. 

35. Saltin, B.: Circulatory Response to Submaximal and MaXImal Exerc1se After 

Thermal Dehydration. J. Appl. PhYSIOl., vol. 19, 1964, pp. 1125-1132. 

36. Senay, L. C., Jr.: Movement of Water, Protein and Crystalloids Between 

Vascular and Extravascular Compartments 1n Heat-Exposed Men DurIng Dehydrat10n 

and FollowIng Lim1ted Relief of Dehydration. J. PhYS10l. (London), vol. 210, 

1970, pp. 617-635. 

17 
37. Senay, L. c., Jr.; and Christensen, H. L.: Changes in Blood Plasma DurIng 

Progressive Dehydration. J. Appl. Physiol., vol. 20, 1965, pp. 1136-1140. 

38. Stein, H. J.; Eliot, J. W.; and Bader, R. A.: Physiological Reactions to Cold 

and Their Effects on the Retention of Acclimatization to Heat. J. Appl. 

Physiol., vol. 1, 1949, pp. 575-585. 

39. Harrison, H. H.; Graveney, H. J.; and Cochraue, L. A.: 

in the Calculation of Relative Change in Plasma Volume. 

Physiol., vol. 50, 1982, pp. 13-21. 

Some Sources of Error 

Eur. J. Appl. 

40. Wlntrobe, H. H.: The Size and Hemoglobin Content of the Erythrocyte. J. Lab. 

Clin. Hed., vol. 17, 1932, pp. 899-912. 

41. Streeten, D. H. P.; and Thorn, G. W.: Use of Changes in the Hean Corpuscular 

Hemoglobin Concentration as an Index of Erythrocyte Hydration. J. Lab. Clln. 

Hed., vol. 49, 1957, pp. 661-671. 

42. Costill, D. L.; Branam, L.; Eddy, D.; and Fink, W.: AlteratIons In Red Cell 

Volume Following Exercise and Dehydration. J. Appl. Physiol., vol. 37, 1974, 

pp. 912-916. 

43. Costill, D. L.; and Fink, W. J.: Plasma Volume Changes FollOWIng ExerCIse and 

Thermal Dehydration. J. Appl. Physiol., vol. 37, 1974, pp. 521-525. 

44. Van Beaumont, W.; and Rochelle, R. H.: Erythrocyte Volume Stability WIth 

Plasma Osmolarity Changes in ExerCIsing Man. Proc. Soc. Exp. BioI. Hed., 

vol. 145, 1974, pp. 240-243. 

45. Eisenman, A. J.; MacKenZIe, L. B.; and Peters, J. P.: ProteIn and Water of 

Serum and Cells of Human Blood, With a Note on the Heasurement of Red Blood 

Cell Volume. J. BIOI. Chem., vol. 116, 1936, pp. 33-45. 

46. Lloyd Jones, E.: On the VarIations in the SpeCIfIC GraVIty of the Blood In 

Health and Disease. J. PhYSlOl. (London), vol. 8, 1887, pp. 1-14. 

47. PhIllips, R. A.; Van Slyke, D.; Hamilton, P.; Dole, V. P.; Emerson, K.; and 

Archibald, R. H.: Measurement of Specific GraVIties of Whole Blood and Plasma 

by Standard Copper Sulfate Solutions. J. BioI. Chern., vol. 183, 1950, 

pp. 305-330. 

48. Van Slyke, D. D.; Hiller, A.; Phlllips, R. A.; Hamllton, P. B.; Dole, V. P.; 

Archibald, R. H.; and Eder, H. A.: The Estimation of Plasma Protein Concentratlon From Plasma Specific GraVIty. J. BIoI. Chern., vol. 183, 1950, 

pp. 331-348. 

18 
49. Holzer, H.; Pogglitsch, H.; Hinghofer-Szalkay, H.; Kenner, T.; Leopold, H.; and 

Passath, A.: Die kontinuierliche Messung der Blutdlchte wahrend der Hamodlalyse. Wiener Klln. Wochenschr., vol. 91, 1979, pp. 762-765. 

50. Kenner, T.; Moser, M.; and Hinghofer-Szalkay, H.: Determination of Cardlac 

Output and of Transcapillary Fluid Exchange by Continuous Recordlng of Blood 

Denslty. Basic Res. Cardiol., vol. 75, 1980, pp. 501-509. 

51. Kenner, T.; Moser, M.; Hinghofer-Szalkay, H.; and Mohl, W.: Indlrect 

Determination of Fluid Filtration and Reabsorption In the Microcirculatlon of 

the Myocardium. Blomed. Tech. (Berlin), vol. 29, 1984, pp. 108-116. 

52. Hlnghofer-Szalkay, H., Martensen, I., and Vogel, G.: Contlnuous Recordlngs of 

the Mass Denslty of Lymph: Introductlon of a New Method. Pfluegers Arch., 

vol. 392 S: R 10, 1982. 

53. Leopold, H.; Hinghofer-Szalkay, H.; Kenner, T.; and Holzer, H.: Rapld 

Determlnatlon of the Sedlmentatlon Rate of Blood Free From the Influence of 

Gravlty. Blomed. Tech. (Berlln), vol. 23, 1978, pp. 99-103. 

54. Hlnghofer-Szalkay, H.; Leopold, T.; Kenner, T.; and Holzer, H.: tiber den 

thermlschen Ausdehnungskoefflzlenten des Blutes und selner Komponenten. 

Blomed. Technlk (Berlln), vol. 25, 1980, pp. 151-157. 

55. Holzer, H.; Leopold, H.; Hlnghofer-Szalkay, H.; Stubchenkirchner, H.; and 

Maurer, E.: GesamtelwelSbestlmmung im Serum durch Dichtemessung nach der 

Blegeschwingermethode. J. Clin. Chern. Clin. Blochem., vol. 16, 1978, 

pp. 391-395. 

56. Hlnghofer-Szalkay, H.; and Wurm, H.: Der Einflus von Hamolyse auf hochgenaue 

Dichtemessungen an Kaplilarblutplasma mlttels der Blegesonwingermethode. J. 

Clin. Chern. Clin. Biochem., vol. 18, 1980, pp. 615-619. 

57. Hinghofer-Szalkay, H.: Volume and Density Changes of BIologIcal FlUIds WIth 

Temperature. J. Appl. Physlol., 1985. In press. 

58. Hinghofer-Szalkay, H.: Fluld and Proteln Shlfts After 'Postural Changes In 

Humans. Am. J. PhYSIOI.: Heart Circ. Physlol., 1985. In press. 

19 
TABLE 1, Variability (X tSE) of Evans blue space (plasma volume) in 

normal males (m, 18-32 yr) and females (f, 30-41 yr) in the basal 

state in 3- tQ 21-day periods, [A] ~ XD aYn - XD aY1 

SubJ. Day 1 Day 4 Pay 8 Day 14 Day 24 Day 28 

12 (m) 3531 3556 [ ... 25] 

±BO ±19 

17 (JII) 3489 3461 [-28) 

±76 ±Bl 

19 (m) 3112 3115 [+3] 

t120 ±105 

7 (m) 3453 3459 [+6] 3422 [ -311 

±160 ±157 ±172 

23 (m) 2971 2989 [+18] 

±97 ±90 

1 (f) 2678 2683 [+5] 

±98 ±91 

20 
TABLE 2. Derlvatlon of the hemoglobin-hematocrit equation for calculation of change In plasma volume 

BaSlc equations: where: PV = plasma volume 

ev = corpuscular volume 

(i) PV = BV - ev BV = (total) blood volume 

Hct = hematocrit, whole 

(i1) ev = BV x Hct body (red + whlte) 

Hb = blood hemoglobin 

concentration 

a = after fluid Shlft 

b = before fluld ShIft 

. Hbb (Ill) BVa = BVb x Hb 

a 

(assumlng unchanged 

Fcell ratIO) 

PV

a - PVb llPV = -;;.;.......-- PVb 

(fraction of PVb) 

(eq. i) llPV = 

(BVa - eVa) - (BVb - evb ) 

BVb - eVb 

BV - (BV x Hct

a ) - [BVb - (BVb x Hctb )] 

(eq. il) llPV a a 

= BVb - (BVb x Hctb ) 

BV (1 - Hct

a ) - BVb(l - Hctb ) 

llPV a 

= BVb(l - Hctb ) 

( eq . 111) llPV 

BVb x (Hbb/Hba )( 1 - Hct

a ) - BVb(l - Hctb ) 

= BVb(l Hct ) c 

(Hbb/Hba )( 1 - Hct ) - (1 - Hct ) Hbb - Hct 

a b 

= 

a llPV - Hctb 

= Hb

a 

x - Hctb 

% hPV • 100 [::: • ~ ~ ::~:]- 100 (Eq. 7) 

21 

- 1 
N 

N 

SUBJECT H. H-S 

1051 

~ 

ci 1050 

'l... 

~ 

~ en 1049 Z 

w 

Q 

Q 

o 

o 

... 1048 CD 

1047 

~ 

o 

SITTING 

t 

10 

STANDING (900 ) 

20 30 

~ 

-

. . 

. 

. 

6----

40 

SUPINE (00 ) 

TIME, min 

50 60 

FIgure 1.- Continuous blood density recordIng In a man sItting, standing, 

and lying supIne. The dIscontInuIty (arrows) occurred when the line 

was flushed wIth heparInized isotonIc sodium chlorIde solution (density: 999.5 gil at 37°C). The flow rate was 1.5 mllmln, and the 

sample volume In the oscIllator was 0.1 mI. 

t 

70 

STANDING (900 ) 
1 Report No 2 Government ~OII No 3 ReciPIent's Catalog No 

NASA TM-86834 

4 Title and Subtitle 5 Report OIIte 

PLASMA VOLUME METHODOLOGY: EVANS BLUE, HEMOGLOBIN- November 1985 

HEMATOCRIT,AND MASS DENSITY TRANSFORMATIONS 6 Pwformlng OrglnlHtIOll Code 

7 Author(s) 8 Performing OrglnllatlOll Report No 

J. E. Greenleaf and H. Hinghofer-Szalkay 85406 

10 Work Unot No 

9 Performing Organozatlon Name and Address 

Ames Research Center 11 Contrlet or Grant No 

Moffett Field, CA 94035 

13 Type of Report and Period Covered 

12 Sponsoring Agency Name and Address Technical Memorandum 

National Aeronautics and Space Administration 14 Sponsoring Agency Code 

Washington, DC 20546 199-21-12-07 

15 Supplementary Notes 

Point of Contact: J. E. Greenleaf, Ames Research Center, MS 239A-1, 

Moffett Field, CA 94035, (415)694-6604 or FTS 464-6604 

16 Abstract 

Detailed descriptions of methods for measuring absolute levels and 

changes in plasma volume are presented along with derivations of pertinent 

equations. Reduction in variability of the Evans blue dye dilution technique using chromatographic column purification suggests that the day-today variability in the plasma volume in humans is less than ±20 mI. Mass 

density determination using the mechanical-oscillator technique provides a 

method for measuring vascular fluid shifts continuously for assessing the 

density of the filtrate, and for quantifying movements of protein across 

microvascular walls. Equations for the calculation of volume and density 

of shifted fluid are presented. 

17 Key Words (Suggested by Author(s)) 18 DlStrobutlon Statement 

T-1824 Microcirculation Unclassified - unlimited 

Blood density Posture 

Plasma density Exercise 

Specific density Subject category - 52 

19 Securoty a_,f (of thIS report) 20 Security o-f (of thiS ~, 21 No of PIgIS I 22 Price' 

Unclassified Unclassified 24 A02 

"For sale by the National TechnlCiI Information StrvlCl, Springfield, Vlrglnl. 22161 
End of Document