THE AIR TURBINE HEMATOCRIT FOR MEASUREMENT OF T H E
RELATIVE VOLUME OF PACKED R E D CELLS*
MAX M. S T R U M I A , M . D . , AND L U I G I A. P R I N C I P A T O , M . D .
W I T H THE TECHNICAL ASSISTANCE O F V I R G I N I A M. S I M S
From the John S. Sharps Research Laboratory of the Bryn Mawr Hospital and the Laboratory
Pennsylvania.
of Clinical Pathology of the Bryn Mawr Hospital, Bryn Mawr,
Among commonly employed procedures in the laboratory of clinical pathology are the estimation of the concentration of hemoglobin, the erythrocyte
count and the hematocrit determination of the relative volume of packed red
cells. The necessity of making a large number of these determinations in a relatively short period of time often greatly reduces their accuracy. Even when they
are done most carefully, considerable variations occur. These may be due to the
method of sampling, to the technic employed and to inaccuracy of the pipets
and colorimeters. In a subject having normal red cells (i.e., cells of normal size,
shape and hemoglobin content) the tests mentioned above are to a certain extent
interchangeable. Partly because of this interchangeability, it is of great interest
to determine which of the tests mentioned yields the most reproducible results.
The use of the air turbine for the determination of the relative volume of the
red blood cellsf (air turbine hematocrit) when carried out on properly collected
double-oxalated blood, yields constant results in a short time. The method was
described in 1943 by Parpart and Ballentine2 who used the apparatus developed
by Beams.1 Unfortunately this procedure is not well known. The purpose of this
paper is to describe the technic and to compare values obtained with the air
turbine hematocrit with those obtained by other means of determination of the
relative volume of packed cells and with data from red cell count and hemoglobin estimation. The effect of sampling on the results of these tests will be
discussed in a separate paper.
A P P A R A T U S AND
TECHNIC
The apparatus (Figf 1) was constructed by J. Russell Mycock.t Capillary
tubes of uniform bore, 0.5 mm. O.D. and 32 mm. long are employed.** The
blood is collected from a suitable vein, preferably the median cephalic, without
stasis. If it is necessary to apply a tourniquet to enter the vein, a short interval
of time is allowed between removal of the tourniquet and withdrawal of blood.
* Presented at, Uic Twenty-Eighth Annual Meeting of the American Society of Clinical
Pathologists, in Chicago, on October 13, 1949.
Received for publication, October 10, 1949.
t The cumbersome terminology, "hematocrit reading of the relative volume of packed
red cells", has been replaced in parts of this report, particular!}' in the illustrations, by the
simple designation, " h e m a t o c r i t " .
t D e p a r t m e n t of Biology, Princeton University.
** The capillary tubes may be obtained from Mr. James G. Graham, Glass Blowing
D e p a r t m e n t , University of Pennsylvania.
419
420
STRUMIA AND
IUUNCIPATO
As an anticoagulant double oxalate is used in concentration of 150 mg. for 100
ml. of blood.* This concentration of double oxalate does not change the volume of
red cells. A concentration of 75 mg./lOO ml. of blood is not sufficient to prevent
coagulation, and a concentration of 300 mg./lOO ml. of blood causes some
shrinkage (see Fig. 2). Heparin as an anticoagulant has not given satisfactory
results.
It is important to keep the blood well mixed by rotating the slanted tube while
filling the capillary tubes with blood. We have found short test tubes (35 mm.
F I G . 1. T h e Air Turbine Hematocrit
x 15 mm. O.D.) satisfactory for the purpose. The capillary tubes are filled with
oxalated blood to about § to f of the total length. The capillary tube is immediately flame-sealed. To obtain a perfectly flat inside surface on the bottom of
the tube, the capillary is sealed while gently rotating it in a very hot flame.
T h e head carrying the tubes measures 10.5 cm. in diameter and 8 mm. in thickness.
P a r p a r t and Ballentine 2 have pointed out t h a t between 20,000 X G and 190,000 X G, the degree of packing approaches an asymptotic value, and t h a t between 40,000 X G and 190,000
X G the red cell volume is reduced by only 0.7 per cent. We have found t h a t it is practical
* The solution
h y d r a t e , 0.6 Gm.
ml. in each tube
concentration of
of double oxalate is prepared as follows:0.4 Gm. potassium oxalate monoammonium oxalate monohydrate, distilled water up to 100 ml. Place 0.3
and dry in oven. This amount, when mixed with 2 ml. of blood, gives a
150 mg./lOO ml.
r
AIR TURBINE HEMATOCRIT
•
•..
421
and safe to use an air pressure of 120 lb./sq. in. (8.4 Kg./cm.2).f The relation between aitpressure and speed is shown in Figure 2. The speed of the rotor was measured with the use
of a General Radio Strobotac.f When the head and capillary tubes are two-thirds filled,
the radius from the center of the liquid column to the center of the head is 3.7 cm. This
radius was used in calculating the relative centrifugal force values, which represent the
HEMATOCRIT
43.0
>
1 1 1 1 1 11
<»
{ DEFIBRINATED BLOOD CONTROL
^
(5
)
i
42.8
'
42.6
i
<i
>
\ i1
•
I\
\
42.4
\
\
42.2
\
•
\ ' 1
\
\
42.0
41.8-
11
11
4 1 4
100
mmg
200
OXALATE
300
400
% (w/v)
FIG. 2. Effect of oxalate concentration on the air turbine hematocrit values of defibrinated blood.
^
V
>
>
average centrifugal forces in the liquid column. The relation between air pressure and
R.C.F.** is shown in Figure 3.
The time required by the column of packed red cells to reach equilibrium at 19,350 r.p.m.
and at 20,600 r.p.m. is shown in Figure 4. The relative centrifugal force at these speeds is
f An air compressor with a 1£ H.P. motor, capable of a maximum pressure of 200 lb./
sq. in. (14 Kg./cm.2) and with a tank of SO gallons capacity (ca. 303 liters) will deliver a
sufficient blast for the proper operation of the air turbine.
| Manufactured by the General Radio Co., Cambridge, Massachusetts.
** R.C.F. = relative centrifugal force expressed as the computed number times gravity.
The approximate formula for calculating the value of the R.C.F. is .0000111 X n2 X 1 gram
X r where n = revolutions per minute and r = distance in centimeters from the point in
question to the center of the centrifuge shaft.
422
STRUMIA AND PRINCIPATO
15,400 X G and 17,400 X G respectively. At 19,350 r.p.m. constant values are obtained in
four minutes. When the speed is increased to 20,600 r.p.m., there is an additional packing,
the constant being reached in one minute. To obtain a very sharp line of separation between red cells and leukocytes it is desirable to centrifuge at about 14,000 r.p.m. (60 lb./sq.
RELATIVE CENTRIFUGAL FORCE:
X GRAVITY
1000
<L<L
/
20
/
14
12
10
8
6
/
/
4
2
20
40
AIR PRESSURE
60
80
100
120
140
lbs./sq. in.
FIG. 3. Relation between air pressure and the relative centrifugal force developed
bv the air turbine.
in. or 4.2 Kg./cm. 2 air pressure) for two minutes, followed by four minutes at about 20,000
r.p.m. (120 lb./sq. in. air pressure). The tubes must be placed vertically immediately after
removal from the centrifuge. Reading of the length of the column can be made by direct
comparison under a dissection microscope with a carefully calibrated steel scale having
0.5 mm. divisions. The use with the dissecting microscope of a movable stage with a 0.1
vernier permits better readings. For accurate measurements we have used a micrometer
AIR T U R B I N E
423
HEMATOCRIT
slide cathetomoter,* measuring down to .001 mm. Such refinement is not necessary in routine work. The column of leukocytes is computed as part of the plasma volume.
•
19,350 RPM
HEMATOCRIT
44
43.5
43
20,600 RPM
^ ^
,42.5
2
3
a
10
II
12
MINUTES
F I G . 4. Relation of time and packing of red cells at various speeds, (a) R a t e of packing
of erythrocytes at 19,350 r.p.m., (b) rate of packing of erythrocytes centrifuged for seven
minutes at 19,350 r.p.m. and for three additional minutes at 20,600 r.p.m.
COMPARATIVE DATA O B T A I N E D W I T H
T H E AIR T U R B I N E H E M A T O C R I T , T H E
WIN-
TROBE HEMATOCRIT, T H E ESTIMATION OF T H E HEMOGLOBIN CONCENTRATION
-
A N D T H E E R Y T H R O C Y T E C O U N T I N V E N O U S D O U B L E - O X A L A T E D BLOOD
All the subjects were healthy, young adults. All specimens were obtained
from the arm vein, with the subject recumbent and the arm held horizontally
along the lateral mid-line. All blood samples were obtained without stasis, and
2 ml. of blood Avas mi.xed with 3 mg. of double oxalate (150 mg./lOO ml.). All
determinations from the same sample of blood were carried out within four
hours of collection.
* Manufactured by Cacrtncr Scientific Corporation, 1201 Wrightwood Avenue, Chicago,
Illinois.
424
STRUMIA AND PRINCIPATO
Air turbine hematocrit. The air turbine hematocrit determinations were made
by two independent workers, each using two capillary tubes and making independent readings. The spread of values in Figure 5 represents all four readings
thus obtained.
Wintrobe hematocrit. Wintrobe hematocrit determinations were carried out by
two experienced technicians, each using one tube. The tubes were carefully
capped during the centrifugation, which was carried out for 45 minutes at 3000
r.p.m. Readings were made immediately after centrifugation and the spread of
values in Figure 5 represents the two values obtained.
Hemoglobin determinations. Hemoglobin determinations were made by two
technicians, each using for the transfer a standard pipet with a tolerance variation of less than ± 3 per cent. Each technician made two transfers of blood into
two tubes. The amount of blood used was 20 cu. mm. and the diluent was 5 ml.
of 0.1 per cent sodium carbonate solution. The carbonate solution was measured with an automatic pipet with a tolerance of less than ± 2 per cent. Both
technicians made two readings independently on the two samples using the
Klett photoelectric colorimeter with filter #54. If the two readings agreed
within 0.2 Gm., the values were averaged and the results noted. If the variations
were greater, a third transfer was made with the same pipet and this third value
Avas averaged with the nearest one of the two previously obtained. This work
was carried out by the two workers independently. The spread of the hemoglobin values, therefore, indicates the variation between the average of two
selected readings carried out by two technicians. The values are thus more nearly
accurate than values that would be obtained if a routine determination were
made by a single technician with a single pipet.
Erythrocyte counts. Erythrocyte counts were obtained by two hematology
technicians using pipets with a tolerance for accuracy of less than ± 3 per cent
for the principal interval. Each technician filled one pipet and routinely counted
the red cells on 5 squares of the standard counting chamber. If the values thus
obtained for any of the squares counted by one technician varied more than
10 per cent, another counting chamber was filled from the sa.me pipet and the
count repeated. This is our standard way of counting erythrocytes. The count
was carried out independently by the two workers and the results shown in
Figure 5 represent the spread between these two values.
In Figure 5 the linear values have been so chosen as to be roughly to scale,
as follows: 1 Gm. of hemoglobin/100 ml. = 2.5 hematocrit points = 300,000
erythrocytes/cu. mm.
It can be readily seen that minimal variations were obtained with the air
turbine hematocrit: the maximal variations, in two instances, were respectively
0.6 hematocrit point or 1.4 per cent (#13) and 1.6 per cent (#25). 3
With the Wintrobe method the hematocrit shows greater variations; maximal variations in two cases were two full hematocrit points or 4.2 per cent
( # 8 ) and 5 per cent (#22), respectively. In ten instances the variations were
greater than 2 per cent.
With the estimation of the hemoglobin concentration, the variations were
ERYTHROCYTES
MILLIONS
i //ccuu mm
1
_LJ_
r-
_
i
—-
—
HEMOGLOBIN
g/100 m
7.6
1
1
|
6.8
t=
—
—
ri
1
=
—
—
—
r~
1
HEMATOCRIT
-
:?
- e
L
•
•
p
U
iT B
"
H
1
1-
I
J-
1
K
I
4J
c
•
•
9
MEN
ALL
SUBJECTS
10
II
12 13
14
IS 16
HEALTHY
18
WOME N
»
ARE
17
YOUNG
19 20 21 22
m
L
23 24
B
AIR TURBINE
•
WINTROBE
r
•
25
1
ADULTS
FIG. 5. Comparison of data obtained with the erythrocyte count, the hemoglobin estimation and the hematocrit measure (air turbine and Wintrobe methods) in 25 healthy
young adults. The vertical bars represent the highest and lowest determinations.
425
426
STRTJMIA AND PRINCIPATO
larger, as much as 1.3 Gm. of hemoglobin or 10 per cent (#19). In four instances the variations were greater than 5 per cent.
The maximum variation found in the erythrocyte counts is 540,000/cu. mm.
or 12 per cent (# 19). There are two other instances of variations greater than
300,000 cells (7.6 per cent, # 3 and # 12).
The air turbine hematocrit gives values which average 2.2 per cent less than
the Wintrobe hematocrit, when both procedures are conducted as indicated
previously.
COMPARISON OF DATA OBTAINED BY AIR TURBINE HEMATOCRIT, HEMOGLOBIN
CONCENTRATION AND RED CELL COUNT ON THE SAME SAMPLE OF
DOUBLE-OXALATED BLOOD
This experiment was carried out to determine the relative reproducibility of
the following laboratory determinations: hematocrit, when performed with the
air turbine method, hemoglobin determination using the Klett Colorimeter and
the red blood cell count. Five workers experienced with the air turbine hematocrit determination and five trained hematology technicians carried out this exTABLE 1
COMPARISON OF V A L U E S OP HEMOGLOBIN, ERYTHROCYTE C O U N T S AND HEMATOCRIT
R E A D I N G S B A S E D ON 10 D E T E R M I N A T I O N S M A D E BY 5 W O R K E R S U S I N G THE SAME
SAMPLE OF OXALATED BLOOD
HEMOGLOBIN VALUES
Standard Deviation....
Coefficient of variation.
0.296 Gm. per 100 ml.
2.0L
RED CELL COUNTS
AIR
TURBINE
HEMATOCRIT
VALUES
71.024 per cu.mm.
1.54
0.116
0.27
periment. The workers in the two groups were not identical except that one of
them made determinations with all tests. Each of these workers made two determinations, obtaining the blood from the same sample of double-oxalated fresh
blood. During collection all samples of blood were mixed in an identical manner
and were kept in continuous motion. The pipets employed for hemoglobin determinations were calibrated to contain 20 cu. mm. of blood (the blood being
diluted with 5 ml. of sodium carbonate solution). These pipets and those used
for red cell counts had the tolerance for accuracy previously mentioned. Each
worker employed the same pipet for both determinations. The results are given
in Figure 6. In Figure 6 the value 0 is the average of all ten values obtained. The
ten individual determinations are arranged above or below as per cent variations
from the "normal" average values. Thus, for the red cell count, maximum variations were —3.04 to +2.10 per cent; for the hemoglobin-determination —3.35
to +2.79; for the air turbine hematocrit —0.55 to +0.43 per cent (Table l ) .
F I G . 6. Comparison of d a t a obtained with the air turbine hematocrit, hemoglobin estimation and red cell count by several observers on the same specimen of oxalated blood.
PERCENT VARIATIONS
FROM AVERAGE
«-»
o
<•-
,>-
ZZZZZZZZZZ??
^~~~~~~~~~JL~H
JZZZZZZZZZZZZZZZZZZZZZ
j!
((
o
IZZZZZZZZZJLZZZZZZZZ";!
[
lr-
4 1
II
•«
IUUI
"ZZZZZZZZZZZZZZZZZZZZZ
————————————
._
ii
HEMATOCRIT
(AIR TURBINE)
HEM0GL08IN
427
REO CELL
COUNT
428
STRTJMIA AND PRINCIPATO
Numerous other experiments carried out on defibrinated blood and on venous
oxalated blood and the routine use of the air turbine as an hematocrit have conclusively shown the reproducibility of results obtained.
CONCLUSIONS
The advantages of the air turbine hematocrit as a means of measuring the
relative volume of packed red cells may be listed as follows:
1. The apparatus is simple and inexpensive, provided that a proper supply
of compressed air is available.
2. The complete test requires about ten minutes, including the time to draw
the blood. •
3. The results are readily reproducible with a moderate amount of technical
skill. A variation of ±0.5 is readily achieved;3 the variations with the Wintrobe
hematocrit in a comparable series of tests are about five fold (±2.5 per cent);
in the hemoglobin estimation and the red cell counts the variations are still
larger, ± 5 per cent and ± 6 per cent, respectively, even when tests are conducted under optimal conditions. In routine tests the variations are at least
twice as large.
REFERENCES
1. B E A M S , J. W., AND W E E D , A. J . : Scientific a p p a r a t u s and laboratory methods; a simple
u l t r a centrifuge. Science, 74: 44-46, 1931.
2. PARPART, A. K., AND B A L L E N T I N E , R . : Hematocrit determination of relative cell volume.
Science, 98: 545-546, 1943.
3. STRUMIA, M . M . , W A L L , R., AND STRUMIA, P . V . : A m e t h o d for estimation of blood
volume. Am. J. Clin. P a t h . , 19: 483-487, 1949.