CA1063381A - Method and apparatus for deriving oxygen association curves - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
An apparatus and method for deriving an oxygen associa-tion curve for a blood sample wherein the sample is placed on a transparent support and is covered by a gas-permeable membrane element. The support is mounted in a gas treatment chamber with transparent windows on opposite sides of the support to provide an optical path through the support normal to the sample.
Radiant energy is directed along this optical path, said radiant energy including two light frequencies, one having a wavelength at which there is substantially no change in absorbance as bet-ween oxygenated and deoxygenated blood and the other having a wavelength at which there is a relatively large change in absorbance as between oxygenated and deoxygenated blood. A
controlled source of deoxygenating gas, such as nitrogen, and a controlled source of oxygen are connected to the chamber.
An oxygen electrode is mounted in the chamber and generates the X component, corresponding to oxygen in the chamber, in an X-Y
recorder. The difference in absorption of the two frequencies is measured and from this is derived a signal forming the Y
component in the X-Y recorder, which thus provides a curve corresponding to the light absorbance changes in the sample while it is being oxygenated.

Description

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This invention relates to methods and apparatus for :,.;: ,:
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 , . ."~ , .
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. ' -- 4 -- .
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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, ;
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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 O ~ ._ , .
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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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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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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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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 ~- -' ~b~ - 10 -.

~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

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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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Claims (18)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An assembly for use in a photometer for measuring oxygen association curves, comprising a chamber, means for transmitting a measuring light beam through the chamber, light-transmitting cell means arranged to support a thin flat layer of sample material in the chamber in the optical path of the measuring light beam, a source of deoxygenating gas controlled conduit means connecting said deoxygenating gas source to said chamber, a source of oxygen, and controlled conduit means connecting said oxygen source to said chamber.

2. The assembly of claim 1, and wherein said cell means comprises a light-transmitting body having a shallow flat sample-receiving recess and a gas-permeable transparent membrane secured on the body over and covering said recess.

3. The assembly of claim 1, and wherein said cell means comprises a light-transmitting body having a shallow flat sample-receiving recess approximately 0.010 inch in depth, and a gas-permeable transparent membrane secured on the body and covering said recess.

4. The assembly of claim 1, and wherein the chamber is provided with a restricted gas escape passage in a wall portion of the chamber.

5. The assembly of claim 1, and wherein said cell means comprises a light-transmitting body, a trans-parent supporting element mounted on said light-transmitting body and arranged to receive the sample material, and a transparent gas-permeable membrane member adapted to overlie the sample material.

6. The assembly of claim 5, and wherein said light-transmitting body is formed with seat means shaped to receive the transparent supporting element.

7. The assembly of claim 6, and wherein the transparent supporting element projects from opposite sides of said seat means.

8. The assembly of claim 5, and wherein said chamber is provided with an extensible closure member and said light-transmitting body is supportingly connected to said extensible closure member.

9. The assembly of claim 8, and wherein said closure member comprises a vertical plate and wherein the chamber is provided with means slidably supporting said plate for horizontal extension relative to the chamber.

10. The assembly of claim 1, and a light source forming said measuring light beam, said source containing at least two wavelengths, said wavelengths having different absorption characteristics when transmitted through a blood sample undergoing oxygenation, and means to compare the relative absorbances of said two wavelengths with the amount of oxygen admitted into the chamber.

11. The assembly of claim 10, and wherein the means to compare said relative absorbances with the amount of oxygen admitted into the chamber comprises means to derive a first electrical signal in accordance with said relative absorbances, means to derive a second electrical signal in accordance with the amount of oxygen admitted into the chamber, and means to plot said first signal against said second signal.

12. A sample cell assembly for measuring oxygen association curves of hemoglobin comprising a light-trans-mitting 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 said blood sample supporting means.

13. The sample cell assembly of claim 12, and wherein said recess has a depth of the order of 0.010 inch for supporting a thin flat layer of blood in the recess.

14. The sample cell assembly of claim 12, and wherein said body has a reduced portion, and a ring-shaped supporting bracket member supportingly receiving and surrounding said reduced portion.

15. A method of measuring the oxygen association curve of a blood sample comprising arranging the blood sample in the form of a thin flat layer, first deoxygen-ating 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 said flat layer while it is being exposed to oxygen at said controlled rate.

16. The method of claim 15, and wherein said deoxygenating gas comprises nitrogen.

17. The method of claim 15, and continuously plotting the measured light absorbance changes against the amount of oxygen to which the flat layer is being exposed, to thereby derive an oxygen association curve for the blood sample.

18. The method of claim 15, and wherein the flat layer of blood sample is exposed to the deoxygenating gas and to the controlled rate of oxygen via a gas-permeable membrane.