Note: Descriptions are shown in the official language in which they were submitted.
633~
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This invention relates to methods and apparatus for
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measuring the oxygenation characteristics of blood or other
material whose light-absorbing characteristics change while
being treated with a reagent, and more particularly to a
method and apparatus for deriving an oxygenation curve for a
whole blood sample.
The oxygen binding curve (commonly called the "oxygen
dissociation curve") for hemoglobin is formed by the measure-
ments of the fraction of total hemoglobin that is oxygenated
as a function of the partial pressure of vxygen (P02) to which
the hemoglobin sample is exposed. The entire curve and/or
~; parameters derived from it are of substantial physiological
and clinical significance. Currently used techni~ues in this
field employ dual wavelength photometry, and such techniques
comprise passing time-shared measure and reference beams ~M and
through the sample while it undergoes oxygenation and
~¦ utilizing the differences in absorption of these beams as
',~!'.,;,~ measured by a photomultlplier tube and associated circuitry
for deriving the desired "oxygen dissociation curve," which is in
fact an oxygen association curve.
The present inventlon encompasses a method and apparatus
for deriving oxygen association curve lnformation from a blood
sample. In its apparatus aspect, the lnvention relates to an
assembly for use in n photometer for mea~uring oxygen associatlon
curves, comprising a chamber, means for transmitting a measuring
light beam through the chamber, light-transmitting cell means
nrranged to support a thln f:Lat layer of samp:le material in
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the chamber in the optical path of the measuring light beam,
`, a source oE deoxygenating gas, controlled conduit means
connecting the deoxygenating gas source to the chamber, a source
of oxygen, and controlled conduit means connecting the oxygen
source to said chamber.
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In its method aspect, the invention relates to a
method of measuring the oxygen association curve of a blood
sample comprising arranging the blood sample in the form o~
a thin flat layer, first deoxygenating the sample by exposing
it to a deoxYgenating gas, then exposing the deoxygenated -
flat layer of blood sample to oxygen at a controlled rate,
and measuring changes in light absorbance of the flat layer
while it is being exposed to oxygen at the controlled rate.
In a further aspect, the lnvention relates to a
sample cell assembly for measuring oxygen association curves
of hemoglobin comprising a ligh~-transmitting supporting body -
formed with a shallow recess, transparent means on the body
for supporting a blood sample in the recess, and a gas-
permeable transparent membrane member adapted to overlie a
blood sample on the blood sample supporting means. `
The technique and apparatus of the present lnvention
offer numerous advantages, among which are the following:
a. It permits use of undiluted whole blood~ thereby
avoiding possible nonphysiological artifacts.
b. It accomplishes deoxygenation of the blood sample
without tlsing harsh reagents, such as dithionate.
c. It requ:Lres only a very small sample volume, whlch
is lmportant :Ln pedlatric cases and Eor research on rare
hemo~lob:tns.
d. It provides a linear measure of fraction oxyhemoglobin ;
over the co~plete range of oxygenation, unlike reflectance ~,
mensurement technlques.
e. It generates continuous curves and provides for
control and variation of P02 which is uncomplicated, reliable
and which requires only small quantitites of compressed gases.
f. It employs an oxygen-sensing electrode which is
inherently stable and has a long liEetime, and which is not in
direct contact with the blood sample.
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In drawings which illustr~te embodiments of the
invention,
Figure 1 is a diagrammatic vertical cross-sectional
view taken through a typical blood sample oxygenation
association measurement assembly constructed in accordance
with the present invention,
Figure 2 is an enlarged fragmentary horizontal plan
view of the sample cell employed in the assembly, said view
. being taken substantially on the line 2-2 of Figure 1,
Figure 3 is a vertical cross-sectional view taken ,~
substantially on the line 3-3 of Figure 2,
Figure 4 is a fragmentary horiæontal plan view similar
to Figure 2 but showing a modified form of sample cell
according to the present invention,
Figure 5 is a vertical cross-sectional view taken
substantially on the line 5-5 of Figure 4~ ,
Figure 6 is an end eievational view of another
modified form of blood sample oxygen association measurement
assembly in accordance with the present invention,
.20 Figure 7 is a vertical cross-sectional view taken
substantially on the line 7-7 of Figure 6 and diagrammatically
showing associated optical and electrical compon'ents used
with the assembly in this form o the inventi.on,
Pigure 8 is a top plan view of the blood sample supporti.llg
member employed in the embodiment of Figures 6 and 7, said
view being.taken substantially on the line 8-8 of Figure 7, '~
Figure 9 is an elevational view o~ the sample supporting
member taken substa,ntially on the line 9-9 of Figure 7, and
Figure 10 is'an enlarged fragmentary vertical'cross- '
:30 sectional.view taken substantially on the line 10-10 of
Figure 8. '
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3~63381
The techni~ue of the present-inYention in~olves the
use o~ a sample cell wherein a thln film of blood is exposed
to controlled PO2 via a gas-permeable membxane, and th~ough
which simultaneously optical absorption spectroscopic measure- ;
ments are performed. A film thickness of blood o~ 0.010" or
less is employed to permit rapid oxygen exchange within the
blood and to make the undiluted blood sample sufficiently
transparent to permit the optical absorption measurements.
The film thickness must be stable for stable optical measure- -
ments, and must not contain occluded bubbles, for both optical
and gas exchange reasons.
Referring to the drawings, 11 generally designates an
apparatus for deriving oxygen association curves in accordance
with the present invention. The apparatus 11 comprises a gas
chamber 26 of suitable opaque material, such as aluminum or the
like, adapted to be mounted, for example, in the path of the
time-shared monochromatic beams ~M~ ~R~ of a dual wavelength
spectrophotometer. In the typical apparatus illustrated in
Figure 1 of the drawings, the chamber has a transparent window
20 12 in its bottom wall for admitting the time-shared beams ~M~ `
~R~ and has another transparent window 13 in its top wall verti-
cally aligned with window 12 to define an optical path there-
between, the emerging beams being directed toward the photo-
multiplier tube 42 o~ the spectrophotometer.
Designated generally at 14 is a blood sample cell which
is horizontally mounted in the path of the time-shared mono-
chromatic beams ~M~ ~R~ Referring to ~igures 2 and 3, the
aell 14 comprises a generally circular body 15 of suitable
transparent material, such as txansparent plastic material, ;
3~ concentrically formed
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i338~
in its top face portion with a shallow circular recess 16 haviny
a depth of approximately 0.010 inch and a diameter of the order
of 3/8 inch. Respecti-ve inlet and outlet capillary tubes 17 and
18 extend vertically and sealingly through diametrically oppo-
site portions of the body 15 and communicate with corresponding
diametrically opposite portions of recess 16. The body 15 is
formed with a rounded-off rim portion 43 leading to a peripheral
groove 19 which receives the marginal portion of a transparent
gas-permeable membrane 20, said marginal portion being sealing-
ly and clampingly secured in the groove 19 by a resilient O-ring
21, with the main portion of the membrane tightly stretched over
the recess 16.
The gas-permeable transparent membrane 20 is about 0.001
inch thick and may comprise pure silicone rubber film, or suit-
able commercial transparent gas-permeable membrane, such as
Perflex (Trade Mark) OM-110 ~ilicone rubber copolymer, manufac-
tured by Union Carbide Corp., Moorestown, New Jersey.
The body 15 is reduced at its lower portion, as shown at
22, and the reduced lower portion is supportingly received in
a bracket ring 23 provided with a radial supporting arm 2g which
is riqidly secured to the adjacent side wall 25 of chamber 26.
Ring 23 is provided with a set screw 27 diametrically opposite
arm 24 which clampingly secures the reduced lower portion 22
in the ring 23.
The capillary tubes 17 and 18 extend sealingly through side
wall ~5 of the gas chamber 26. ~n oxygen supply conduit 28 pro-
vided with a suitable control valve 29 extends sealingly through
the opposite side wall 30 of chamber 2~, and a nitrogen supply
conduit 31 similarly provided with a suitable control valve 32
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extends sealingly through said side wall 30. Rotatably and sub-
stantially sealingly supported in side wall 30 is the shaft 33
of a fan 34, suitably driven by an external electric motor 35.
A conventional oxygen-sensing electrode 36 is sealingly mounted
in wall 30 and extends into the chamber, the electrode being ex-
ternally connected to suitable circuit means for generating the
X component of a conventional X-Y recorder. The oxygen-sensing
electrode 36 may be similar to Model5331, manufactured by Yellow
Springs Instrument Company, Yellow Springs, Ohio, of the type
known as a "Clark Electrode" (Trade Mark).
A water-absorbent humidifying wick member 46, which can be
readily moistened, is mounted in the lower portion of the chamber
26, for examp~e in a recess provided therefor in the lower por-
tion of wall 25. The wick 46 furnishes the required humidity to
prevent the excessive drying out of the oxygen electrode 36 and
blood sample. The chamber 26 is temperature controlled, to main-
tain a substantially constant temperature therein by means of an
electric heater 37 secured to the chamber in heat-transmitting
relation thereto, whose energization is controlled in a conven-
tionai manner by a temperature-sensing element 40 embedded in the `~
chamber wall adjacent the heater.
The upper portion of wall 25 is provided with a restricted
vent pa~sage 41. In a typical embodiment, the wa}l thickness of
the chamber was about 0.6 inch, and the vent passage 41 was about
0.025 inch in diameter.
In the assoclated dual wavelength spectrophotometer various
wavelength pairs may be employed for the AM and ~-R monochromatic
beams, for example, 650 nm-800 nm, 547 nm-560 nm or 650 nm-724 nm.
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^` 1063~
The advantages of employin~ a dual ~avelength system of measuxe-
ment include a) automatic correction for any changes in scattered
light during the generation of a test curve, b) use of a
re~erence beam path that also goes through the sample, and
c) the potential capabllity o~ measuring hematocrit and other
parameters, as we~l as generating the oxygen association curve,
at the same time. ;~
A single wavelength measurement system may be employed
with the gas chamber and blood cell apparatus of the present ;~
~0 invention, providing reduced cost and greater simplicity of ~`
construction, but not providing automatic correction for possible
changes in scattered light during the generation of the oxygen
association curve.
In operation, the membrane cell 14 is supplied with a
sample of whole blood, admitted therein through the inlet
capillary tube 17. The oxy-deoxy transition is started in
this enVironment, with the chamber 26 being first filled with
N2 (including about 5% C02) through conduit 31 by opening valve
32, whereby to deoxygenate the blood sample. The initial
deoxygenation of the sample requires an exposure of about 15
minutes. Thereafter, ~ith valve 3~ closed and the spectro-
photo~eter system in opexation, an oxygen binding curve
~oxygen association curve) i8 generated Oll the associat~d
X-Y recorder b~ ope~ating valve 29 to slowly introduce 2
~including about 5% CO2). The oxygen supply system may include
a syringe pump ox other pump to provlde a rate of oxygen input
~ufPicient to achieve 20% oxygen in the chamber in 5 to 10 min-
utes. The variation of PO2 with time will be exponential rather
than linear, so that the oxygen binding cur~e is prefexably
generated using the X-Y recorder, with the oxygen electrode 36
driving the X axis and the PM cube 42 providing the signals
for generating the Y component in the recorder. Since the
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~ 6338~
oxygen electrode 36'does not come in contact with the blood sample,
the PO2 in the chamber must be varied sufficiently slowly that
the blood is always effectively in equilibrium with the P02 in , '
the chamber.
The dimensions of fan 34 are chosen to provide uni~orm
vigorous mixing during the test run.
The 0.010 inch blood layer in the cell 14 permits con- ' ,
trolled oxygenation of the blood, as above described, within -
5 to 10 minutes, with the initial deoxygenation requiring about ~ '
10 15 minutes. By employing a lesser blood layer thickness (for '
example, 0.004 inch or less) the required exposure times may , ,
be correspondingly reduced.
Mounting the cell 14 horizontally, as above described,
eliminates possible difficulties associated with settling o~ red
blood cells during long experiments.
The blood sample is pretreated with suitable reagent,
such as Heparin, or other well known anti-coagulant, to prevent
clotting. This is done at the time of dra~Jing the blood from
the patient.
Figures 4 and 5 show another form of membrane cell,
designated generally at 14', in accordance with the present
invention. The cell 14' comprises a stainless steel ring 15'
formed with an annular internal seat 50 in which is cemented
a circular quartz window Sl whose top ~ace is approximately
'0.010 inch below the top plane of the xing, to def,ine the
main blood sample-receiving recess. Diametrically opposite ''
channel recesses 52 and 53 are formed in the top portion of
the ring, communicating with said main recess. The capillary ~ '
' tubes 18 and 17 extend ~ertically and sealingly through the
opposite portions of the ring and communicate with the channel
~ecesses 52 and 53. The ring has the lower reduced portion 22'
which is clampingly secured in the supporting ring ?3 in the ,
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63381
same manner as previously described in connection with Figures
2 and 3.
Ring 15' has the rounded-off top rim portion 43' leading
to a peripheral groove 19', and a stretched transparent gas-
permeable membrane 20 is secured over the blood sample recess
by means of an O-ring 21 clamping the marginal portion of the
membrane in the g~oove 19', as in the previously described
cell 14.
Various means may be employed, other than by use of an
O-ring, for securing the stretched transparent gas-permeable
membrane over the blood sample recess. Thus, for example,
the membrane may be clamped in a suitable jig assembly. A film
of silicone rubber cement may be applied to the rim portion of
the main cell body and this rim portion may be clamped against
the membrane, stretching it taut, and held thereagainst until
the cement is c~red, after which excess membrane materiàl may
be cut away.
While thè above-described procedure employs whole blood
as a sample, it is also possible to employ diluted blood or
hemoglobin in solution as a sample in said procedure.
In so~e cases it is possible and desirable to omit the
covering membrane 20 and merely employ a thin layer of blood o~
the order of O.OOl inch thick on a flat transparent supporting
plate, suitably mounted in the chamber 26 in place of celll4.
Under these aonditions, care~ul control of the humidity in the
chamber is necessary.
In the embodiment illustrated in Figures 6 to 10, the
gas chamber, shown at 26' i9 provided with a large circular
aperture 60 in its right end wall 25', as viewed in Figure 7. `~
30 A vertical cover plate 61 is slidably supported on a pair of ;~
parallel horizontal, relatively long support rod members 62, 62
threadedly secured respectively in the lower CGrner portions ~- -
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~L0633~31
of end wall 25', said rod members being pro~ided with outer head
portions 63, 63 to limit the rightward extension of cover plate
61 to the dotted view position thereo~ shown in Figure 7. Cover
plate 61 is formed with the integral inwardly projecting circular
boss 64 shaped to substantially fit in the aperture 60 in the
closed position of the cover plate. The sup2ort rod members 62,
62 are provided at their inner end portions with annular detent
grooves 65 and the bottom edge portion of cover plate 61 is pro-
vided with spring-biased detent balls 66 yieldably and lockingly
engageable in the grooves 65 to hold the cover plate 61 in
closed position, as shown in full-line view in Figure 7. Cover
plate 61 is provided externally with a central operatiny knob 67.
Rigidly secured to the central portion of boss 64 is
the horizontal supporting arm 24' which carries the blood sample
cell element shown at 68. The supporting cell element 68 com-
prises an originally annular opaque member having a bottom central '~
aperture 69 and an upstanding peripheral flange 70, with opposite ;
cut~away lats 71, 71, as shown in Figure 8 to provide finger
access for removing samples, as will be presently described.
As will be further described, the blood sample is supported
on a circular transparent disc 72, of glass, or the like, placed
in the sèat defined by element 68, and is covered by a gas- ,
permeable membrane disc 73 placed over the sample and held
thereon by the sur~ace tension o~ the ~lood sample, shown at
74 (see Figure 10). ,
The dual wavelength optical system employed may be
similar to thak previously described, but a more economical
~ystem which may be employed comprises a suitable polychromatic
light source 75 containing the reference wavelength ~R and the
measure wavelength ~M~ arranged so as to provide a beam 76
directed upwardly through the bottom window 12, the aperture 69,
the blood sample 74 between the discs 72 and 73, and through
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~L~63381
the top window 13. The beam 76, after absorption by the blood
sample, passes thxough the top window 13 to a beam splitter 77,
such as a half-silvered 45~ mirror, and forms two respective
exit be~ms 78 and 79, the beam 78 being transmitted through
the half-silvered mirror and the beam 79 being reflected there-
from. Beam 78 passes through a suitable ~R filter 80 to a
first phototube 81 and beam 79 passes through a suitable ~M
filter 82' to a second phototube 82. The output currents I~
and I~M of the phototubes 81 and 82 are applied to the respective
inputs of a conventional computing logarithmic amplifier 83,
providing an output signal equal to log IAM ~ log I~R, or log
~M
I~R
The out,put signal of amplifier 83 is delivered to the Y
input of the associated ~-Y recorder. As in the previously
described embodiments of the invention, an oxygen-sensing `'
electrode is provided in the gas chamber 26' for generating the
X component of the recorder. '
In a typical arrangement according to Figures 7 to 10, ,, '
20 the glass disc 72 has a diameter of approximately 18mm. and ''' ,
the transparent membrane disc is about 10 mm. in diameter. The
supporting cell element 68 is shaped to conformably receive ~i~
the disc 72 in the manner shown in Figures 7 to 10, where it ' `' '
will be seen that portions'o disc 72 project. outwardly beyond
the flats 71, 71 to enable them to be easily grasped between ,' ,
the operator's fingers when it is desired to lift the disc 72,
carrying the blood sample 74 and the membrane disc 73, off
the holder 68 (with the cover plate 61 in its open dotted
view position of Figure 7). ~;
The sample blood layer to be tested is prepared as
follows: a disc 72 is placed in 'the holder 68. A drop of ,''
blood of 1-2 micro liters in volume is placed on the central
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1~363381
portion of disc 72. The membrane disc 73 (which may be com-
posed of General Electric MEM-213 material, manufactured by
General Electric Co., Inc., Schenectady, N. Y., or equi~alent)
is placed over the blood sample, which then forms a thin layer
74 by capillary action combined with the weight of the disc.
After this the cover plate 61 is moved to its closea position,
shown in full-line view in Figure 7.
The blood layer 74 has a thickness of less than 25
microns, and may be as small as between lO and 20 microns in
thickness. This permits complete deoxygenation in 1.5 to 2.0
minutes, rapid one-step oxygenation in 3 to 5 seconds, and pro-
duction of equilibrium oxygen association curves in about 5
minutes. By contrast, a blood layer 0.01 lnch thick requires ~`
from 15 to 20 minutes for deoxygenation and 30 to 50 seconds
for rapid one-step oxygenation. The use of the gas-permeable,
non-porous membrane disc 73 also retards water loss and permits
safe handling of the blood layer in ambient air.
The appropriate wavelengths for dual wavelength spectro-
photometry where a blood layer of 25 microns or less ln thickness
is used are 438 nm and 448 nm, and therefore these wavelengths
are preferably employed for AM and ~.
With the sample installed in the holder 68 in the manner
above described, it is first precycled for proper blood condition~
ing ~a highly lmportant re~uixement), as ~ollows: the chamber 26'
i5 rapidly flushed with nitrogen containing 5% CO2, for example, ;
~or about one m~nute at a xate of 100 cc per minute, to deoxy-
gen~te the sample, The sample is then oxygenated rapidly by
~dm~tting ox~gen cont~ining 5% CO2 for about 5 seconds at about
120 mm pressure. The X-Y xecorder is then calibrated by ad~ust-
ing it so that the optical signal provides a Y-component reading
,
which is stored as representing lO0~ oxygenation. The chamber
~6' is then purged with nitrogen containing 5% CO2. After
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~6~63381
deoxygenation, the opti~al signal is again read and stored as
0% oxygenation. The two stored values are used to set the zero
offset and scale expansion to the Y axis of the X-Y recorder
so that the graph will automatica~ly span 0-100% on the Y axis.
i The oxygen association curve for the sample may then be
derived b~ following the procedure previously described, namely
by admitting oxygen into the chamber and measuring the change
in log I~M against oxygen concentration, using the ahove- -
I
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described optical system in cooperation with the x-Y recorder.
This gives a measurement of the fractional change in optical
density that corresponds to the fraction of oxyhemoglobin in
the sample over the range of oxygen concentration in chamber 26'.
The technique herein described produces a blood layer
that is relatively resistant to e~aporation and quite stable with
time, so that, for example, repeated association curve measure-
ments can be made on the same sample.
As will be apparent, when a sample is to be removed from
the chamber 26', the cover plate 61 is pulled open to its fully
20 extended position, allowing the operator to easily remove the `
sample by graisping the projecting opposite edge portions of disc
72 between his fingers and lifting the disc out of the holder 68.
While certain specific embodiments of improved methods
and apparatus Eor deriving oxygen association curves for blood
samples have been disclosed in the foregoing description, it
will be understood that various modifications within the spirit
of the invention may occur to those skilled in the art. There- `
fore, it is intended that no limitations be placed on the ;
29 in~ention except as defined by the scope of the appended claims.
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