T H E APPLICATION OF T H E GRAVIMETRIC TECHNIC TO T H E
SIMULTANEOUS DETERMINATION OF PLASMA VOLUME USING
RADIOIODINATED HUMAN SERUM ALBUMIN AND R E D CELL
MASS USING SODIUM RADIOCHROMATE
R O B E R T E . 551PF, M.D., J O E M. W E B B E R , M . D . , AND G. R I C H A R D G R O V E , P H . D .
Department
of Research, Miami
Valley Hospital,
Dayton,
Ohio
The application of the gravimetric technic23 to the measurement of red cell
mass with sodium radiochromate tagged erythrocytes provides simplicity and
accuracy that answer the requirements of routine determinations in the clinical
laboratory. The gravimetric technic for the estimation of plasma volume may
be readily coupled with the determination of red cell mass in order to provide
accurate values for total blood volume and total body hematocrit.
Gray and Sterling 9,10,2l reported the successful tagging of erythrocytes with
radioactive chromium-51 in 1950. The use of sodium radiochromate has replaced
the radioisotopes of iron, 6,12 phosphorus, 11,16 potassium,3 carbon monoxide,"
and most other methods for direct determination of red cell mass. Gray and
Frank 6 , 8 in 1953 described the use of radioactive chromic chloride as a substance
for tagging plasma, and they presented a method for simultaneous estimation
of red cell mass and plasma volume, using the isotope of chromium exclusively.
The method described here eliminates the complex separations of plasma, and
washings and drying of erythrocytes, and utilizes the venous hematocrit reading
determined by radioiodinated human serum albumin (RISA) in order to compute the specific activity of the red blood cells tagged with chromium-51. The
scintillation-well counter, projection analytical balance, and standard solutions
of copper sulfate for determining specific gravity remain as essential tools in
this procedure. The use of concentrated ACD (C-ACD) solution makes possible
the completion of the tagging of erythrocytes, injections, and sampling in approximately 35 minutes.
MATERIALS AND
METHODS
Preparation of ACD solution for tagging erythrocytes. An ACD solution containing disodium citrate (monohydrate) 2.5 mg., citric acid 0.8 Gin., and dexRoceived, J a n u a r y 10, 1956; revision received, February 2; accepted for publication
February 6.
Dr. Zipf is Director, Department of Research, and Associate Pathologist, Miami Valley
Hospital; Dr. Webber is Resident in Pathology and Fellow in Research, Miami Valley
Hospital; Dr. Grove is Consulting Nuclear Physicist, D e p a r t m e n t of Research, Miami
Valley Hospital, and Physics Group Leader, Mound Laboratory, Monsanto Chemical
Company, Miamisburg, Ohio.
This work was supported in part by grants from the Montgomery County Society for
Cancer Control, and was developed in conjunction with the United States Air Force, R e search Contract AF 33(616) 2756, Torrence G. McGuire, M.D., C a p t . , USAF (MC), ProjectResearch Officer. T h e authors wish to acknowledge the invaluable technical assistance of
M a r y C. Paige, Lois S. Chiles, Irene G. Gilleland, Helen L. Hudson, and Dolores Mowry.
4S7
488
ZIPF ET AL.
Vol. 26
trose 1.2 Gm., in 100 ml. of physiologic saline solution provides an excellent
medium for the tagging of chromium-51 to red cells. The C-ACD solution is
made by adding the above quantities of substances to 10 ml. of physiologic saline
solution so that 0.1 ml. of C-ACD solution may be used to tag 5 ml. of blood.
Studies have indicated that this C-ACD solution accelerates the binding of
chromium-51 to the red cells, producing virtually complete tagging in 20 minutes.
Preparation of vials for tagging erythrocytes. An aliquot of 0.4 ml. of C-ACD
solution is placed in an empty 30-ml. rubber-capped vial. The vial and its rubber
cap are then autoclaved at 120C. for 15 minutes. The unused vials are sterilized
again in 14 days.
Procedure for simultaneous determination of plasma volume and, red, cell mass.
An aliquot of stock solution of RISA, containing 20 no. of activity, is drawn into
a 20-ml. syringe, and, at the same time, 100 nc. of radiochromium are introduced
into a "tagging vial." This is clone prior to the initial venepuncture. Then 20
ml. of blood are removed, gently added to the "tagging vial," and evenly mixed.
The 20 ^c. aliquot of stock solution of RISA is then injected into the vein of the
subject, utilizing the needle already in place.
Tagging of the red cells is complete after 20 minutes, at which time 17 to 19
ml. of the tagged blood are drawn into a clean sterile syringe, and 2 to 4 ml. are
transferred to a screw-cap vial23 for the preparation of standard solutions of
chromium. A sample (Sample A) of about 10 ml. of blood is then withdrawn
from a site other than that of injection, and placed in tubes containing heparin,
again with the needle in place. The tagged blood is injected intravenously
through the sampling needle. Under normal conditions a mixing time of 10 to
15 minutes is allowed to elapse, and a final sample (Sample B) of 10 ml. of blood
are obtained from another vein (or a different site) and placed in the heparintubes.
Determination of -plasma volume and venous isotope hematocrit reading. The
plasma volume and venous isotope hematocrit reading are determined by the
gravimetric method23 utilizing blood from Sample A.
Preparation of chromium-51 standard and, determination of red, cell mass. The
sample of tagged blood taken for the preparation of the standard solution of
Cr-51 is gently inverted 20 to 40 times in order to obtain maximum mixing, and
a single drop is placed in a previously weighed vial, and the mass of the drop is
determined. The drop is then diluted to approximately 1.0 ml. by the addition
of physiologic saline solution in order to obtain a standard geometry in the well
counter. The specific activity of the chromium-tagged whole blood is then determined as counts per minute per milliliter of whole blood. The remaining blood
is centrifuged at 3000 r.p.m., and the specific activity of the plasma is determined
by using a single drop of plasma in a similar manner. The specific activity of the
red cells can be calculated from the following measured quantities:
Hi = the venous isotope hematocrit reading determined from the
values with RISA;
Sp = the specific activity of the plasma in the "tagging vial";
S n = the specific activity of the whole blood in the "tagging vial";
May
1956
PLASMA VOLUME DETERMINATION
4.S9
sp = the specific activity of the plasma in the withdrawn blood;
sB = the specific activity of the withdrawn whole blood;
D = the dose (in ml.) of tagged blood injected.
Thus, the specific activity of the injected red cells is:
SB -
SR
SP(1
- H.)
H,
However, inasmuch as 0.5 to 0.6 ml. of saline are present in the "tagging vial,"
the hematocrit reading of the tagged blood will be lowered by approximately
1.0 per cent if 20 ml. of blood are added and the hematrocrit reading is between
35 and 55 per cent. For values of the hematocrit less than 35 per cent, the change
due to the presence of these solutions has progressively less significance. The
hematocrit reading may be corrected by reducing the venous isotope hematocrit
reading by approximately 1.0 per cent.
The whole blood sample, B, is gently inverted 20 times and approximately 1
ml. of the mixed blood is placed in a previously weighed vial (by means of a
Pasteur pipet), and the weight of the blood is determined. The specific activity
of the whole blood is then determined in counts per minute per milliliter. A similar
sample of plasma from sample B is obtained, and the specific activity of the
plasma is determined. The specific activity of the red cells from this sample is
calculated from the following equation:
8,1 _ s„ - s,.(l - Hi)
m
•
Thus, the red cell volume is given by:
v
,r x
S B - S r ( l ~ Hi) X Hi
SB
— Sr(l — Hi)
the true blood volume by:
V B (Cr, I) = V K (Cr) + V P (I),
and the whole body hematocrit by:
H =
VK(CT)
Vn(Cr) + VH(I) '
RESULTS
The average values for the volumes of plasma, red cells, and total blood (calculated from RISA and sodium radiochromate) on 45 normal male persons are
presented in Table 1. The average values are expressed in milliliters per kilogram
of body weight as a normalizing factor. The values obtained for 2 subgroups and
for the entire group are listed in Table 1. Group I included 25 college athletes,
weighing from 77.3 to 107.0 kg., with an average weight of 87.8 kg. The second
group was composed of 20 non-athletes, weighing from 62.7 to 95.0 kg., with an
average weight of 75.6 kg. A comparison of the average values of the red cell
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Z I P F ET
Vol. 26
AL.
TABLE 1
N O R M A L I Z E D V A L U E S O F W H O L E BLOOD ( V B ) , PLASMA (Vp), AND R E D C E L L
VOLUME ( V R ) IN M I L L I L I T E R S PER KILOGRAM OP B O D Y W E I G H T
RISA
(ml./kg.)
Description
Group I (25 athletic men)
Group II (20 non-athletic men)
Groups I and II (45 persons)
* V„ (Cr,
Rachromate
(ml./kg.)
Combined
(ml./kg.)
VB
Vp
VR
VB
VP
VR
V B (Cr, I ) '
70.6
71.7
71.2
37.5
38.7
3S.1
33.1
33.0
33.0
66.4
64.2
65.3
35.2
34.1
34.6
31.2
30.1
30.6
68.7
68.S
6S.7
I) is the sum of V P ( R I S A ) and
V K (Cr-51).
mass, relative to the chromium-51 values, obtained by the 2 methods reveals
a difference of 6.1 per cent for the combined groups. These RISA and chromium51 values of VR were determined independently, and they indicate the difference
in body and venous hematocrit readings which has been reported by other investigators. 15 ' 19 ' 20 In the case of the whole blood and plasma volumes, the values
are not independent because the specific activity of the samples of plasma in
both methods is proportional to the venous "vers-hematocrit" (defined as unity
minus the hematocrit reading, or the ratio of the plasma volume to the whole
blood volume). Thus, as described in the procedure, the dilution with RISA
provides a direct determination of plasma volume, whereas the tagged erythrocytes permit the direct measurement of the red cell mass. The sum of these 2
determinations provides an accurate value of the total blood volume. These
values are also listed in Table 1, and they are essentially the same for both groups.
The average value for the 45 individual deviations of red cell volumes, as determined by the 2 methods, was also calculated and found to be 8.2 per cent.
This agrees well with the deviation of the average values of 7.8 per cent that
were calculated from Table 1. The difference in total body and venous hematocrit
readings may be the result of the presence of differential pools of plasma within
the body. However, work by Allen1 and Gregersen" indicates that the extra
plasma may be distributed throughout the body, and possibly the plasma is not
present as discrete anatomic pools. Perhaps the difference in the values of VR
found by the 2 methods results from a differential in the velocity of flow of plasma
and red blood cells, associated with the progressive capillary restriction in the
arterial side of the vasculature. Studies by Gitlin and Janeway 7 and Wasserman,
Karlmann, and Mayerson22 seem to indicate that a significant degree of extracirculatory diffusion of albumin might be partially responsible for this phenomenon. From these conjectures it seems that a satisfactory explanation for
the difference between body and venous hematocrit readings must be further
elucidated.
Accurate values for the circulating total blood volume and red cell mass can
be determined by RISA alone, if sufficient time is allowed for complete mixing
of the injected activity, as is required for conditions such as splenomegaly or
congestive heart failure. Relatively accurate values of the total blood volume
can be obtained from the RISA plasma volume by using the isotope value of
May
1956
PLASMA VOLUME DETERMINATION
491
the venous hematocrit reading and the ratio of the whole body hematocrit
reading to venous hematocrit reading. This is very nearly the ratio of the average
values of the red cell volumes determined by chromium-51 and EISA, respectively (Table 1). Thus:
NK UJ
1 - 0.921-1, N p U ;
and
V B *(I) = V K *(I) + V P ( I ) .
The values listed in Table 1 were calculated to have a standard probable error
of approximately 8 per cent for the distribution of normal individual values
around the average values. Inasmuch as the number of persons in this study was
not especially large, the histogrammatic distribution of the values did not define
the normal values to a precision commensurate with the precision of the method.
However, it was noticed that some of these cases resulted in high values. This was
especially true in the instance of sodium radiochromate, probably because of
incomplete injections of the tagged cells.
mscussroN
Studies on human beings by Berson,3 and on dogs by Reeve19 and Root, 20
have shown that there is a fairly constant disparity between venous hematocrit
readings and total body hematocrit readings. The ratios of total body hematocrit
values to venous hematocrit values have been found to vary from 0.86 to 0.92.
The studies of Reeve, on splenectomized dogs subjected to various forms of artificially induced hemodynamic change, indicate that the relation between venous
and body hematocrit remains constant. The only exception to this finding was
in those animals that were infused with Dextran, in which instances the ratio
fell to levels below 0.80. Berson and Yalow3 reported a large series of cases, including many acute and chronic pathologic conditions in human beings. The
same relation between body and venous hematocrit values was found, although
more variation was noted than in the splenectomized animals. The studies reported in the control subjects used in the development of this method confirm
this relation.
Berson2 observed that at least 2 clinical states may alter the effective circulation or mixing of the tagged erythrocytes. Splenomegaly and congestive heart
failure seem to prolong the time required for uniform distribution of red cells.
Slow circulation through the spleen is thought to exist in splenomegaly, whereas
increased venous pressure and prolonged time of circulation seem to play a major
role in congestive heart failure. Prior to estimation of blood volume with tagged
red cells, 30 to 40 minutes should be allowed for complete mixing in instances
where circulation may be impeded. However, Nomoff16 has shown that relatively
complete mixing of red cells tagged with Cr-51 occurs in the presence of congestive
heart failure, and in conditions that might be expected to prolong complete dis* Indicates the corrected values.
492
Z I P F ET
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Vol. 26
tribution of red cells. This work16 indicates that the findings of Berson,2 and
other investigators who have reported progressively diminishing activity of red
cells in serial sampling, may be the result of dissociation of radiophosphorus and
radiopotassium from the erythrocytes, inasmuch as these isotopes were used
for the determinations of blood volume.
The relative firmness of the radioiodine tag to the albumin, and the strong
bond between the radiochromium and hemoglobin, make possible the determination of survival time of erythrocytes tagged with Cr-51. 4 , 1 7 , 1 8 Repeated injections of RISA may be made if necessary, in order to make interim determinations of blood and plasma volumes. The centrifuge hematocrit must be used in
such instances for the determination of Cr-51 activity of erythrocytes.
The gravimetric technic was shown23,24 to have a standard probable error
of precision of less than 3 per cent. The high degree of precision of determinations of blood and plasma volume by this method increases the significance of
variations from the estimated or predicted normal values. In our institution
this has resulted in clinicians having a high degree of confidence in measurements
of blood, plasma, and red cell mass.
SUMMAKY
1. The application of the gravimetric technic to the simultaneous, routine
clinical determination of plasma volume (using RISA) and red cell mass (using
sodium radiochromate) is described. Concentrated ACD solution may be used
to hasten tagging, without significant alteration of the hematocrit reading of
the tagged sample.
2. The findings of a fairly constant difference between total hematocrit values
and venous hematocrit values is reported and discussed. Certain conditions that
may alter the circulation of the red cells are mentioned.
3. Normal values for young men less than 25 years of age are presented, together with estimations of precision and accuracy of (1) predicted normal values
and (2) actually measured values of volume of blood, plasma, and red cells.
4. The application of the gravimetric technic to the determination of plasma
volume, total blood volume, and red cell mass demonstrates that there is good
correlation between the direct and indirect determination of red cell mass. 15 ' 19 ' 20
This substantiates the reliability of obtaining a clinically dependable value for
absolute red cell mass by appropriate correction of the indirect value for red
cell mass as obtained by using RISA.
SUMMARIO li\T INTEKLIiYGUA
1. Es describite le application del technica gravimetric al simultanee determination clinico-routinari de (1) le volumine de plasma (con le uso de human
albumina serai a iodo radioactive [ASIR]) e (2) le massa de erythrocytes (con le
uso de radiochromato de natrium). Un concentrate solution de ACD pote esser
usate pro accelerar le etiquettage, sin currer le risco de significative alterationes
in le valores de hematocrite pro le specimens etiquettate.
2. Es reportate e discutite le constatation de un satis constante differentia
May 1956
PLASMA VOLUME DETERMINATION
493
inter le valores de hematocrite total e le valores de hematocrite venose. Certe
conditiones capace a alterar le circulation erythrocytic es mentionate.
3. Normal valores pro juvene adultos mascule de minus que 25 annos de etate
es preseiitate, insimul con estimationes del precision e exactitude de (1) le
predicite valores normal e (2) le realmente mesurate valores pro volumine sanguinee, plasma, e erythrocytes.
4. Le application del technica gravimetric al determination de volumine de
plasma, volumine de sanguine total, e massa de erythrocytes demonstra un bon
grado de correlation inter le directe e le indirecte determination del massa erythrocytic. Isto corrobora le justification del principio que clinicamente exacte
valores pro le absolute massa erythrocytic es obtenite per le appropriate correction del valores indirecte que es obtenite pro le massa erythrocytic per le uso
de AS1R.
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