129

J. Physiol. (I95I1) I I5, I29-I42

THE PLASMA IN THE PACKED CELL COLUMN

OF THE HAEMATOCRIT
BY D. LEESON AND E. B. REEVE*
From the Clinical Research Unit, Guy's Hospital, London
(Received 4 January 1951)
When the anticoagulant, applied force, time of spinning and form of haematocrit
tube are standardized, the haematocrit method gives consistent estimates of
the volume of packed cells in a sample of blood. A number of workers have
determined the relationship between this volume and the volume estimated
by an independent method, and it is agreed that the haematocrit packed cell
column contains a small amount of plasma. For the independent method
most workers have used one of two types of procedure, which may be termed
the indirect and the direct. In the indirect procedure the volume of plasma
in a sample of blood is determined. The difference between this volume and
the volume of plasma determined by the haematocrit method is the volume
of plasma trapped in the haematocrit packed cell column. In the direct
procedure the packed cells of the haematocrit are separated from the super-
natant plasma and analysed directly for their contained plasma.
Table 1 summarizes the results of comparisons made by a number of workers
between various forms of the haematocrit method and various forms of the
indirect procedure (Section A) and direct procedure (Section B). It is seen
that the majority of workers' estimates of trapped plasma, expressed as
a percentage of the packed cell column, lie between 3 and 5 %. However,
Maizels (1945), using a haematocrit method which appears to have been
comparable with that used by the majority of workers, found that only about
2-2 % of the packed cell column was plasma, and Chapin & Ross (1942), using
a greater centrifugal force for double the time, found that about 8-5 % of the
packed cell column was plasma.
McLain & Ruhe (1949), from their results on defibrinated ox blood listed in
Table 2, have concluded that many of the independent methods are not
sufficiently accurate to determine the proportion of plasma in the packed cell
column. It is seen that their independent methods of the indirect type gave
* Work undertaken on behalf of the Medical Research Council.
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 INTERCELLULAR PLASMA OF HABMATOCRIT 131
estimates of 4-17-9 % packed cell plasma, and most of these estimates differed
significantly from those obtained with direct independent methods.
Thus, at present, there is doubt about the amount of plasma in the haemato-
crit packed cell coluimn, and the results of McLain & Ruhe suggest that this
is at least in part due to insufficient accuracy of the independent methods
used.
The determination of the true volume of the cells from haematocrit estimates
is of importance for the accurate estimation of total red cell and blood volume
(Reeve, 1948) in which we have been interested. We have therefore developed.
a new independent method of estimating the cell volume of blood samples,
which has advantages over any previously described, and with this we have
determined the amounts of plasma trapped in the packed cell column of
a particular form of the haematocrit method.
TABLE 2. Results reported by McLain & Ruhe (1949) on defibrinated ox blood, using two equiva-
lent haematocrit methods and different independent methods of the indirect and direct
type
Plasma, % of
No. of packed cells
expe. ±S.D. Independent method
A. Indirect form of independent mdhod
13 7-0±4-7 T 1824
13 4-3+2-1 Plasma protein before and Total N - non-protein N
13 4-0±2-2 after dilution with iso- Total N by macro-Kjeldahl
18 9-3±3-3 tonic saline, estimated by Heat coagulable solids
23 x 3 9-0-17-9 Six other methods based on plasma specific gravity determina-
22 x 3f s.D.7-1-10-8 tions before and after blood dilution with isotonic saline
B. Direct form of independent method
15 2-0±1-2 Heat coagulable solids Washed out with 2 vol. saline to
10 3-4±1-0 T 1824 1 vol. packed cells

METHODS
The haematocrit method
The standard haematocrit method we chose was to spin 10 cm. columns of heparinized blood in
3 mm. diameter haematocrit tubes at 3000 r.p.m. for 30 min. The distance from the bottom of the
haematocrit tube to the centrifuge spindle, that is the radius of the centrifuge head, in all experi-
ments was 15 cm. All haematocrit tubes were calibrated. Readings were made by lens with the
tubes held against an anti-parallax scale engraved on both sides of a 3 mm. thick piece of Perspex
sheeting.
The bore of the haematocrit tube. Since a number of workers have used different bores of
haematocrit tubes, we tested if, at constant time and force of spinning, the bore affected the
packed cell volume. Four samples of human and one of rabbit blood were spun in triplicate in
sealed haematocrit tubes of 1, 3 and 9 mm. bore. The results are summarized in Table 3. The
packed cell volumes given by the 3 and 9 mm. bore tubes show good agreement, but average about
1% more cells than those given by the 1 mm. bore tubes.
Time of centrifugation. Some workers have spun their haematocrit tubes for as much as 60 mi.
To test the effect of periods of spinning longer than 30 min., six samples of human and four of
rabbit blood, all heparinized, were spun in triplicate in 3 mm. bore tubes for 30 min., and, after
132 D. LEESON AND E. B. REEVE
reading, for a further period of 30 min. The results are shown in Table 4. A second 30 min. period
of spinning both human and rabbit blood on the average decreased the packed cell volume by
1% of its 30 min. value.

TABLE 3. Percentage packed cell estimates in haematocrit tubes of 1 mm., 3 mm. and 9 mm.
bore spun simultaneously at 3000 r.p.m. for 30 min.
All blood columns were 100 mm. long.
Animal 1 mm. bore 3 mm. bore 9mm. bore*
Man 1 45*2 45-3 45-1
45.3 45.20 45-7 45 60 45.3 45*20
45-1 45X8
2 41 5 42.3 41 9
41 5 41-53 42 0 42-13 42-4 42-15
41-6 42 1
3 43.1 43.3 43-4
43-0 42-97 43-6 43.43 43-3 43 40
42-8 43-4 43.5
4 42-9 43-1 43-1
42-7 42 80 43-0 43 07 43.3 43 03
42*8 43-1 42-7
Rabbit 34-0 34.5 34-6
34-0 34 03 34-2 34-37 34-8 34.57
34-1 34.4 34-3
Means 41-31 41-72 41-67
* The triplicate estimates made in the 9 mm. bore tubes show the greatest scatter. This was in
partibecause the top surface of the packed cell coluimn in these tubes was often irregular and there.
fore the volume of the packed cells was less easy to determine than in the narrower bore tubes.

TABLE 4. The mean percentages of packed cells of human and rabbit blood samples spun in
triplicate in 3 mm. bore haematocrit tubes at 3000 r.p.m. for first 30, and then 60, min.
All blood columns were 100 mm. long
After 30 mi. After 60 min. Differences
Man 1 49.93 49.43 0-50
2 44.57 44-07 0.50
3 43-97 43.53 0-44
4 43.80 43-27 0-53
5 45-37 44*90 0 47
6 43*40 42-93 0-47
Mean 45-17 44-69 0 48
Rabbit 1 37-82 37.37 0 45
2 36-23 35-91 0 32
3 38-29 37-91 0*38
4 40-94 40-62 0 32
Mean 38-32 37.95 0 37

The independent method of estimating the volume of the cells

Serum proteins and bovine albumin were labelled with radioactive iodine.
For the indirect method small quantities of these solutions were added to
heparinized whole blood samples, and the volume of plasma was determined
from the dilution of the radioactivity. For the direct method radioactive
protein solution was added to whole blood samples; these were spun in specially
designed haematocrit tubes; and the radioactivity of measured samples of
packed cells was determined without plasma extraction.
 INTERCELLULAR PLASMA OF HAEMATOCRIT 133
Preparation of radioactive protein. Human and rabbit serum proteins and bovine albumin
(Armour Laboratories) were labelled with 1311 by the method of Banks, Francis, Mulligan &
Wormall (1950, 1951). To a mixture of 0-1 ml. of a freshly prepared solution of KI (1-4 g./100 ml.),
0-05 ml. of 0-1 N-KI03 (0-3567 g. KI03/100 ml.) and 0-05 ml. of 0.1 N-HCl, was added 0-2 ml.
of a carrier-free solution of radioactive NaI (obtained from A.E.R.E., Harwell), containing
50-150 sic. of 1311. This solution of radioactive iodine in KI was added to 'ammoniated' serum
prepared by treating 3 ml. of human or rabbit serum, or of a 7 g./100 ml. solution of crystalline
bovine albumin, with 0-6 ml. of a 1 in 5 dilution of 0-880 ammonia.

Fig. 1. Special haematocrit tube. The tube is made of 9 mm. bore celluloid tubing, is 105 mm.
long and its base is a perforated disc. A perforated cap screws on to the base and holds in
place a rubber disc. During centrifugation the hole beneath the rubber is filled with plasticine
to prevent deformation of the rubber. After centrifugation the plasticine is removed and the
cells are drawn off as illustrated.
After about 20 min. the pH was brought to about 7-4 by the addition of acetic acid, and the
serum was transferred to a dialysing sac and dialysed against ice-cold 0-85% (w/v) NaCl. This
saline was changed 12-14 times over a period of 48 hr., and finally the serum was dialysed for
2 hr. in a boiling tube containing about 30 ml. of saline, and the 131I content of this saline was
determined. It always proved negligible. The labelled protein was then transferred to a sterile
tube and stored in the refrigerator.
Indirect procedure. 2-5 mg. heparin was added to about 30 ml. of blood, which was then divided
into two parts. The first part consisted of two 8 ml. portions, to each of which 0- 1 ml. of radioactive
protein solution was added. After gentle mixing for 5-10 min. about 1 ml. of blood was removed
from each portion to make duplicate haematocrit estimates in 3 mm. bore haematocrit tubes, and
the remainders were centrifuged for 10 min. at 2000 r.p.m. The radioactive (sample) plasma was
then removed from each. The plasma of the second part of the original blood was separated and
two radioactive standards were prepared by adding 0-1 ml. of radioactive protein solution to
two 4 ml. portions of (inactive) plasma. For estimation of radioactivity duplicate 1 ml. volumes
of plasma were taken from each sample and from each standard plasma, and were added to
10 ml. portions of a solution of 0-9 g. NaCl and 1 0 g. tri-sodium citrate per 100 ml. The 131I
contents were estimated in the Geiger-Miiller counter described by Veall (1948).
134 D. LEESON AND E. B. REEVE
Direct procedure. About 30 ml. of heparinized blood were divided into two parts. To the first
part of about 16 ml. was added 1 ml. of radioactive protein solution, and after thorough mixing
this radioactive blood was transferred to two special centrifuge tubes, illustrated in Fig. 1, in
which it was centrifuged for 30 min. at 3000 r.p.m. in a centrifuge head of 15 cm. radius. The
supernatant plasma was then separated and kept; the buffy coat and the top few mm. of packed
cells were discarded. The centrifuge tube was mounted vertically in a clamp, and a needle fitting
tightly to a well oiled 2 ml. 8yringe was pushed through the rubber disc into the bottom of the
packed cell column, and the cells were slowly sucked off till the upper end of the cell column had
fallen to within 5 mm. of the needle tip. In this way approximately the lower 85 % of the packed
cell column was drawn off; it was usually drawn off in two portions corresponding to the lower
third and upper two-thirds of the cells removed.
Standard samples consisted of 0-2 ml. supernatant radioactive plasma, 1 ml. of inactive packed
cells (separated from the second part of the original blood), and 10 ml. of the saline citrate solution.
Packed cell samples consisted of 1 ml. of radioactive packed cells and 10-2 ml. of saline citrate
solution. The red cells in all samples were lysed with saponin before counting. Counts were made
in the Geiger-Muller counter for liquids described by Veall (1948).
Possible causes of error with the iodinated protein methods
In both types of method the amount of plasma in the packed cell column is determined from the
quantity of "III in the plasma of whole blood samples. Errors will arise if free 131J diffuses into
the cells. All samples of iodinated protein have been dialysed till negligible quantities of 's'I
were found in the dialysate. tinder the conditions used here the chief part of the remaining iodine
should be linked firmly to the tyrosine residue of the protein. In all instances but two, precipitation
of the iodinated protein with a mixture of three parts 95 % (v/v) ethanol and one part ethyl ether
brought to the boil removed 99 % of the contained 131I. In these two instances the estimates of
plasma trapped in the packed cell column obtained did not differ significantly from the other
estimates and they have therefore been accepted. We think therefore that this cause of error is
excluded. Errors will also arise if labelled protein is attached to the cells. All human blood
samples were grouped, and the sera used for iodination were chosen so that they did not agglutinate
the cells of the samples to which they were added. Such iodinated serum proteins would not be
expected to become attached to the cells. Dr J. 0. Laws kindly made electrophoretic observations
which indicated that iodinated bovine albumin was not attached to the red cells. Finally errors
will arise if 1II is precipitated during centrifugation with the cells. All iodinated protein solutions
were kept sterile in the refrigerator and used within 3 or 4 days of preparation. Before about half
the experiments the iodinated protein solutions were centrifuged at 1400g for 60 min., after
which only the top layers were drawn off for use.

RESULTS
The percentage of the packed cell column consisting of plasma as
estimated with protein labelled with 131I
Human blood: indirect procedure
Table 5 summarizes the results of duplicate observations on eleven samples
of blood drawn from eight healthy men and women. On different occasions
analyses were made on three samples drawn from R. and two from H. and these
show fair agreement. The labelled serum protein was of the same blood group
as the blood to which it was added. The mean estimate of plasma as the per-
centage of the haematocrit packed cell column is 4-25 + 1-74 (S.D. about mean).
 INTERCELLULAR PLASMA OF HAEMATOCRIT 135
Human blood: direct procedure
Table 6 summarizes the results of observations on nine samples drawn from
healthy donors. In most experiments analysis was made of the plasma content
of the lower third and the upper two-thirds of the packed cell column. In six
experiments labelled serum protein was added to blood of the same group.
In the other three experiments labelled bovine serum albun was added.
The results obtained with the two different protein solutions do not differ
significantly. On the average the lower 85% of the packed cell coluimn, spun
in 9 mm. bore tubes at 3000 r.p.m. for 30 min., contained 5-10 + 0-40 % of
plasma. The lower third of this averaged 4-0 % and the upper two-thirds
5-8 % plasma. Since the lowest levels of the packed cell column are subjected
to the greatest forces, it would be expected that in them packing would be
more complete than in the higher levels.
TABLE 5. The percentage of the packed cell volume of human blood spun under the standard
conditions that consists of plasma, as estimated by the indirect form of the radio-active
method
Duplicate estimates Haematocrit,
of plasma packed cells
expressed as Mean plasma % of
Subject % packed cells % packed cells whole blood
R. 4.7 5'0 4-85 44.4
4-1 4.9 4.5 44-4
6-7 5*0 5*85 45.8
H. 6*1 6*9 6.5 43-3
8.2 5.9 7 05 44-8
G. 2*1 5.7 3.9 47.7
P. 4.4 3-6 4*0 42*2
M. 2*6 4-2 3.4 46-5
W. 3*2 2*6 2*9 49*2
L. -1-2 3.8 1*3 38-2
B. 4-1 1.0 2*55 43.9
Means - - 4-25±1.74 (S.D.) 44-6

TABLE 6. The percentage of the packed cell colujmn of human blood spun under the standard
conditions in 9 mm. bore tubes, that consists of plasma, as estimated by the direct form of
the radioactive method
Plasma, % of Plasma, % of Mean plasma,
lower I packed upper i packed % of packed
Subject cell column* cell column* cell column*
R. 4-0
4-7t 6*3t 5.5
A 3-9 6*4 5.5
G. 4*1 5.5 5-0
M. 40 5-8 5-2
B. 4*6 -
Hu. 4.3 5-8 5.3
Wr. A 3-6 5.5 4-8
Me. A 3-2 5.0 4-4
Means 4-04 5-76 5-10±0-40 (S.D.)
* Approximately the lower 85 % of the packed cell column was drawn off for analysis.
t The plasma in the lower and upper halves of the packed cell column was estimated.
A indicates estimates made with iodinated bovine albuimin.
136 D. LEESON AND E. B. REEVE
Rabbit blood: direct procedure
Since the observations by the indirect and direct procedure on human blood
showed close agreement, observations on rabbit blood were made only with
the direct procedure, Two small modifications of the technique used with human
blood were made. Because our rabbits had an average of about 35 % packed
cells, which gave a rather short packed cell column in the special haematocrit
tubes, the cell content was raised to about 40 % by preliminary gentle
centrifugation with removal af some plasma. No attempt was made to group
the rabbit's blood to which the labelled rabbit serum solution was added.
However, we were careful to draw blood samples from rabbits that had never
been transfused, and on no occasion was there visible haemolysis in the
supernatant plasma of blood to which radioactive serum protein solution had
been added. The results of observations on ten samples of blood drawn from
healthy rabbits, and two from rabbits 3 weeks after 40-50 % of their blood
had been removed, are shown in Table 7. In eight observations labelled serum
TABLE 7. The plasma trapped in the packed cell column of rabbit's blood estimated by
the direct radioactive method
The blood was spun under the standard conditions in 9 mm. bore tubes
Plasma, % of Plasma, % of Mean plasma,
lower * packed upper I packed % of packed
cell column* cell column* cell column* Notes
Normal rabbits
Rabbit 1 3-8 5.3 4-8
2 4-4 6-0 5-4
3 4.9 6-9 6-2
4 50 6-9 6*2
5 40 6-1 5.3
6 4-1 6-0 5-4 Percentage of cells
7 4-1 59 5.3 raised artificially
8 4-2 5-8 5-2
9 A 4-2 6-8 5.9
10 A4-0 5.9 5.3
Mean 4-27 6-16 5-5
±0X46 (S.D.)
Bled rabbits
1 A50 7-6 6-71 40-50% total blood
2 A 5*1 7.3 6-6f removed 3 weeks
earlier
* Approximately the lower 85% of the packed cell colulmn was drawn off for analysis.
A indicates estimates made with iodinated bovine albumin.
protein and in four labelled bovine albumin was used. In the blood drawn
from normal rabbits the average plasma expressed as a percentage of the
lower 85% of the packed cell column was 5-5 + 0-46. The lower third of this
portion of the packed cell column contained on the average 433 %, the upper
two-thirds 6-2 %. The estimates given by the labelled serum protein and
labelled serum albumin agreed. The results, averaging 6-7 %, on the blood of
the two animals that had been bled, differ from the mean of the other restlts
 INTERCELLULAR PLASMA OF HAEMATOCRIT 137
by 2-5 times the standard deviation, and this difference is probably significant.
Further observations are required on blood drawn from abnormal animals.
The effect of a raised red cell sedimentation rate on the amount
of plasma in the packed cell column
The red cell sedimentation rate of three samples of blood drawn from healthy
men was increased by the addition of fibrinogen, and the plasma trapped in
the packed cell column was measured by the direct procedure, using labelled
bovine albumin. The blood with raised sedimentation rate was prepared as
follows: 20 ml. of heparinized blood were gently centrifuged, 5 ml. of plasma
were drawn off, and in their place were added 5 ml. of isotonic sodium chloride
containing 1-7 g. % human fibrinogen. The sedimentation rates were measured
in 10 cm. blood columns in 9 mm. bore haematocrit tubes and, because of the
shortness of the blood column, were considerably underestimated. The results
of these experiments are shown in Table 8. The average plasma expressed as
a percentage of the lower 85% of the packed cell column of blood containing
rapidly sedimenting red cells was 3-6 %, or 1.5% less than in the packed cell
column of untreated blood. The percentage of plasma in the lower third,
averaging 2-8 and in the upper two-thirds, averaging 4'1, show that packing
has been more complete throughout the whole cell column.
TABLE 8. The effect of raised erythrocyte sedimentation rate on the trapped plasma. The estimates
were made as those in Table 6, but the sedimentation rate of the cells was raised beforehand
by the addition of fibrinogen to the blood
Plasma, % of Plasma, % of Mean plasma,
lower * packed upper I packed % of packed Sedimentation
Subject cell column cell column cell column rate, mm./hr.*
R. A 2-9 40 3-6 48
B. A 3-0 4-6 4-0 40
H. A 2-5 3-6 3.3 50
Mean 2-80 4.07 3-63
* Estimated in 9 mm. bore tubes containing 100 mm. columns of heparinized blood.
A indicates estimates made with iodinated bovine albumin.

DISCUSSION
Conclusions from our observations
First, the estimates on human blood made by the indirect and direct radioactive
methods, respectively 4-25 +174% (S.D.) and 5-1 + 0-40%, show good agree-
ment. Second, the observations with the direct method show that less plasma
is trapped in the lower than in the upper parts of the packed cell column.
Therefore, under given conditions average packing will be slightly more
efficient in the short cell column of anaemic than in the long cell column of
polycythaemic blood. Third, under the conditions used here, prolonging the
period of centrifugation by a further 30 min. results in slightly less trapped
138 D. LEESON AND E. B. REEVE
plasma. Fourth, considerable variation in red cell sedimentation rate may
cause some, but not much, variation in the amount of plasma trapped in the
packed cell column. Fifth, our observations suggest that packing is slightly
more efficient in 1 mm. bore than in 3 mm. bore haematocrit tubes.
It should be noted that the direct form of the radioactive protein method
described here has three advantages over previous direct methods: (i) there is
no risk of contamination with supernatant plasma, (ii) washing the plasma
out of the packed cells is uinnecessary and (iii) some haemolysis does not affect
results.
Comparison of various workers' results on human blood
With the exception of the results of Chapin & Ros, the results obtained by
the workers using indirect methods (see Table 1) and haematocrit methods
similar to those used by us, show good agreement, and also agree well with the
observations reported here. Mayerson, Lyons, Parson, Nieset & Trautman
(1948) claim to have confirmed the results of Chapin & Ross. The agreement by
the majority of workers indicates that the results of Chapin & Ross are wrong,
and Barnes, Loutit & Reeve (1948) have suggested a possible explanation.
Of the observations made by the direct method, those of Oberst (1935) were
made primarily to determine the error caused by adherent plasma to the
estimation of the sodium content of the red cells, and the radius of the
centrifuge head is not stated. Maizels (1945) found a significantly lower
content of plasma in the packed cell column than we did. The reason for this
is uncertain, but perhaps he did not succeed in washing all the plasma out of
the cells. The single observation of Gregersen & Schiro (1938) shows good
agreement with ours.
Hence the majority of workers find that 4-5 % of the packed cell column
of human blood spun at 3000 r.p.m. for 30 min. in a centrifuge head of about
15 cm. radius consists of plasma.

Mathematical estimate of trapped plasma

The volume of plasma that would be trapped, supposing the red cells were
rigid flat circular discs and were packed in apposition can be estimated
mathematically. The least and most efficient forms of packing and the- method
of making the calculation are illustrated in Fig. 2. The most efficient form of
packing would result in 9 % of the packed cell column consisting of plasma.
It is clear, therefore, that the cells are not packed as flat discs, but are deformed
by the centrifugal force to fill the intercellular spaces shown in the diagram.
This would be expected from the easy deformation of the red cells that has
been observed in the capillary circulation (Krogh, 1922).
 INTERCELLULAR PLASMA OF HAEMATOCRIT 139
1
Errors of independent methods of estimating cell volume
Indirect procedure. A known quantity, N, of marking agent, which dissolves
in and confines itself to the plasma, is added to a known volume, B, of whole
blood. After mixing, the marked plasma is separated and n, the quantity of
marking agent in unit volume, determined. P, the volume of plasma in B,
is determined from P = Nl/n. The volume of cells in B is B - P. If H is the cell
volume given by the haematocrit, than Q, the percentage of the packed cell
column that consists of plasma, is given by (H ) x 100.

H~~~~~~

r~~~~~~~~~~~~~~~

A B
Fig. 2. Geometrical packing of the cells. A. Least efficient packing. The % intercellular plasma is
4r2 - 7n.2
given by 4r2 x 100 and is 21-5 % of the total volume. B. Most efficient packing. The
% intercellular plasma is given by 2V3r'
-
213r2 x 100 and is 9 % of the total volume.

P may also be determined from the change in concentration of plasma protein

caused by the addition of a known volume of isotonic saline. If V ml. of
isotonic saline are added to the blood sample and the protein concentration
before the addition is A1 and after the addition is A2 mg. per ml., then
P=Vx A2
Al -A2.
Errors in estimating H and B may be neglected since with careful technique
they should be small. The accuracy of the estimate of Q is then chiefly dependent
on two factors, the accuracy of the estimate of P and the size of the haematocrit.
The accuracy of the estimate of P depends on the accuracy of estimation of
the quantities or concentrations N, n, A1 and A2. Assume that these are
measured with an error of ± 1 % of their total quantity or concentration.
Then, in half the cases when the errors do not cancel out, P is determined to
+ 2% of its total volume from N/n and to + 4 % of its total volume from
A21(Aj- A2).
PH. CXV. 10
140 D. LEESON AND E. B. REEVE
As shown by the following examples the size of H influences the error of
estimate of Q. Let P be estimated with an error of + 2 % of its total volume.
1. Let H=52-5, B= 100 and P=50.
P is estimated as 49 or 51.
Qthen=4.7 +2.
2. Let H= 31b5, B = 100 and P= 70.
P is estimated as 68-6 or 71-4.
Q then = 4*7 + 4-4.
Direct procedure. Whole blood, with or without added plasma marking agent,
is spun under standard conditions. M, the mg. of plasma protein or marking
agent in 1 ml. of the supernatant plasma, is determined. The packed cells are
separated and analysed for m, their total content in mg. of plasma protein or
marking agent. If C is the volume of packed cells analysed, then Q, the per-
centage of their volume consisting of plasma, is given by M C If M and m
.

are estimated with an error of 1 % of their total quantities, then in half the
cases when the errors fail to cancel out, the total quantity of plasma contained
in the packed cell column will be estimated with an error of + 2 %. To illustrate,
using the first example given above: let H = 52-5, B = 100 and P = 50.
Then 2-5 ml. plasma is trapped in every 52-5 ml. packed cells.
This volume of 2-5 ml. will be estimated with an accuracy of + 005 ml.
Q will then be estimated as 4-7 + 0 1.
From this analysis it is clear that the direct procedure is inherently much
more accurate than the indirect. With the indirect procedure considerable
errors are particularly liable to occur if blood samples with low packed cell
volumes are analysed and if P is determined from V x A 2A . Examination
of the standard deviations of our results, shown in Tables 5, 6, and 7, and of
the results of other workers shown in Tables 1 and 2, confirms the greater
accuracy of the direct procedure. It is curious that the only workers whose
results with the indirect procedure show a standard deviation of less than
1*0 are Chapin & Ross, and as already seen the weight of evidence indicates
that these results are erroneous.

The findings of McLain & Ruhe (1949)

From their results, summarized in Table 2, McLain & Ruhe concluded that
'most dilution procedures are not well adapted to precise serum volume
estimates. Correction of conventional haematocrit results by a constant factor
based on dilution methods is not justified by present data'. In this paper we
conclude that it is justifiable to correct haematocrit results by a constant
factor, and therefore it is necessary to examine the data on which McLain
 INTERCELLULAR PLASMA OF HAEMATOCRIT 141
& Ruhe base their conclusions. Nine of the twelve independent methods of
estimating packed cell volume used by them were indirect and based on the
change in protein concentration or specific gravity caused by the addition of
a volume of saline about equal to the volume of serum of the blood sample to
which it was added. P was determined from V x A2 . With six of their
A1 - A2
indirect methods they obtained mean values of 9O0-17 9 % plasma in the packed
cell column. The standard errors of the means of these six methods are all
above 7-0 and the results can therefore be disregarded. The mean estimates of
three (one direct and two indirect) of the remaining six methods ranged between
3.4 and 4 3 % and did not differ significantly. One indirect method was based
on the determination of the heat-coagulable solids in small plasma samples,
the direct method giving a mean of 2+ 1-2 %, and the indirect a mean of
9-3 + 3.3 /. Finally the T 1824 indirect procedure gave a mean of 7 0 + 4.7 /.
The standard deviation of their results with this last method was 3-4 times
that of the results obtained with the same method by Gregersen & Schiro (1938)
(on dog blood), Shohl & Hunter (1941), and Morrison (1948), suggesting that
the technique of the latter workers was much more precise than that of
McLain & Ruhe. McLain & Ruhe themselves note that 'precise estimates of
serum volume by the dilution method require the utmost accuracy in
measuring the index concentrations'. We think that McLain & Ruhe have
shown that very variable estimates of the amount of plasma trapped in the
packed cell column are obtained with inaccurate methods, but not that
variable estimates are obtained with carefully tested precise methods.

Standardizing haematocrit estimates

In the past there has been considerable variation in the haematocrit methods
used by different workers. As a result uncertainty has arisen in comparing
the results obtained by one method with those obtained by another, and in
deriving true cell volumes from the various haematocrit estimates. Further,
the interpretation of results has often been made almost impossible because
a number of workers have given insufficient details of the haematocrit methods
they have used. The best method of standardizing haematocrit estimates is to
transform them into true cell volumes. There is now enough evidence to justify
estimating the true cell volume in heparinized blood from healthy subjects
from the packed cell volume x 095, when the packed cell volume is determined
by the haematocrit method used by us. Hence the user of another form of
haematocrit method can determine the correction factor necessary to apply
to the packed cell volume given by that method, by comparing its estimates
with those obtained with the haematocrit method used here, corrected x 0 95.
To avoid uncertainty in interpretation of their results all workers using
10-2
142 D. LEESON AND E. B. REEVE
haematocrit methods should state the anticoagulant used, the dimensions of
the haematocrit tubes, the lengths of the column of blood in them, the radius
of the centrifuge head, the speed and time of centrifugation, the correction
factor applied and how it was obtained.

SUMMARY
1. New direct and indirect methods, using protein labelled with radioactive
iodine, for estimating the amount of plasma trappea in the packed cell column
of the haematocrit are described.
2. With these methods it was found that when 100 mm. columns of
heparinized blood from normal men were spun in 3 mm. bore tubes in
a centrifuge head of 15 cm. radius at 3000 r.p.m. for 30 min., about 5 % of the
packed cell column consisted of plasma. When heparinized blood from normal
rabbits was spun under the same conditions, about 5-5 % of the packed cell
column consisted of plasma. In all bloods the lower layers of the packed cell
column contained less plasma than the upper layers.
3. The quantity of trapped plasma was a little reduced by increasing the
sedimentation rate of the erythrocytes, by spinning in narrower (1 mm.) bore
tubes or by spinning for 60 instead of 30 min.
4. The results of previous workers are reviewed and compared with those
reported here.
We wish to thank Professor Wormall and his colleagues for instructing us in their methods of
preparation of proteins labelled with radioactive iodine.

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