CA1044124A - Micro-gasometric method and apparatus - Google Patents

Abstract

MICRO-GASOMETRIC METHOD
AND APPARATUS

Abstract A method and apparatus for determining the carbon dioxide content of micro samples of blood, serum, or other body fluid. In performing the method, a micro quantity of sample is introduced into one compartment of a double-compartmented open-topped reaction vessel, the other compartment containing a reagent capable of liberating the carbon dioxide from the sample when the two are mixed. The open top of the vessel is then sealed so that its interior communicates only with a capillary manometer, and the contents of the vessel are then thoroughly mixed. The volume of evolved gas is measured directly from the graduated capillary manometer by observing the extent of displacement of an indicating liquid therein. The structure of the device is described, along with the structure and method for adjusting the volume of the reaction vessel, after sealing and before mixing, to locate the meniscus of the liquid in the capillary at a selected starting point.

Description

Background U.S. Patent 3,756~782 issued eo Philips on September 4, 1973 discloses a method and apparatus for determining the carbon dioxide content of a blood sample in which an acid-containing syringe is coupled in gas-tight relation to a vial containing a ; blood sample. The acid is injected into the vial and the sample, upon such acidification, releases carbon dioxide which may be quantitatively measured by the extend of displacement of the syrlnge plunger. As brought out in that patent, such a system is based on the classical Van Slyke procedure but, unlike prior systems used in the clinical laboratory, is relatively uncomplicated in struc-ture and operation. The system of patent 3,756,782 does have a shortcoming, however; it is not well suited Eor determining the carbon dioxide content of ~icro samples (i.e., under 0~5 milliliters) of biological fluid.
Equipment for measuring the carbon dioxide content in micro samples is known, as disclosed, for example, in U.S. Paten-t

2,680,060 issued to Natelson on June 1, 1954. The disadvantages ~, Of available micro-gasometers have already been indicated; in general, they are bulky, expensive, and complex in both structure ; and operation. It will be readily appreciated that operative complexities are particularly undesirable, not simply because they consume time and cause delays, but because they increase the risk ~; of manipulative error which, in any dIagnostic procedure, may have -~
most se~rious consequences. Also, because such devices are some-~ times provided with mercury-containing tubes to measure pressure, ~-; ~ they present additional risks which attend the use oE a hazardous ~ , substance~
Summary This invention is directed to a system for measuring ~, carbon dioxide in biological fluids which is simple and reliable ~, in operation and construction, and which is particularly suited for ehe processing of micro samples of such fluids. For purposes of ss,,~ ~ d ap /1 , "~

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this disclosure, the ter~ "micro" refers to sample volumes less than 0.5 milliliLers and includes volumes which may even be small enough to fall within what is commonly designated the ultra-micro range (i.e., less tha~ 0.05 milliliters). The method and apparatus of this invention are therefore well suited for use in measuring the carbon dioxide content of blood (including serum or plasma) taken from pediatric patients and other subjects from whom the taking of larger samples might have undesirable effects.
One aspect of this invention lies in the discovery that . , ~
the carbon dioxide content of a sample of biological fluid may be ; effectively and readily determined by utili.~ing a two-compartmented reaction vessel in a gas-tight system, the reaction vessel being in direct communication with an open-topped transparent capillary manometer containing an indicating liquid. The separate compart-:;
ments contain measured quantities of the body fluid and a gas-liberating reagent and, on mixing of the fluid and reagent, carbon dioxide is liberated from the sample to cause the indicating liquid in the manometer to be displaced a distance proportional to the volume of liberated gas.
Broadly speaking, the present invention provides a method oE measurlng the carbon dioxide content of a biological fluid, comprising the steps of introducing a measured sample of biological fluid into one portion of an open-topped reaction vessel ~-~
containing in another portion thereof a reagent capable of liberat-ing the carbon dio~ide content of the biological fluid u~on admixture therewith, then sealing the vessel so that the interior thereof communicates only with the lower end of a graduated trans-parent capillary tube containi.ng an indicator liquid defining a ;
meniscus visible through the wall of the tube, then adjusting the volume of the interior of the vessel to shift the indicator liquid in the tube until the meniscus is at a selected position along the graduated capi.llary tube~ and thereafter mixing the contents of the reaction vessel while the same communicates only with the

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capilla~y tube to measure the amount of carbon dioxide liberated by observing the extent of displacement of the indicator liquid in the tube.
The above method may be carried ou~ in a devlce for measuring the carbon dioxide content of a biological fluid comprising a base adapted to support a reaction vessel, an open-topped reaction vessel removably supported upon the base, a mano-meter head mounted upon the base for movement between a raised position spaced from the reaction vessel and a lowered position in sealing engagement with the open top thereof, the head including a transparent capillary manometer tube, the tube communicating at its lower end with the interior of the reaction ~essel when the head is lowered and containing an indicator liquid normally dis-posed at the tube~s lower end, and clamping means for releasably securing the head in sealing engagement with the vessel and for altering slightly the position of the head when lowered to alter the volume of the vessel and thereby adjust the position of the . . , liquid in the tube to a selected reference point.

In the disclosed embodiment of the invention, the :1 . :, ,, reaction vessel may be transparent (or at least translucent~ and have lts compartments arranged in side-by-side relation, divided by an upstanding partition which is lower than the full height of the vessel. The perimetric upper edge or mouth of the open-topped ~essel extends along a single plane and is adapted to make sealing contact with the gasket carried by the manometer head assembly.
Where replacability of the capillary manometer is desired, a second gasket may be provided to insure a gas-tight sealing engage-ment between the capillary tube and the remainder of the manometer ~ .
~' head.
The size oE the base and head are such -that the entire apparatus is easily lifted, tipped, and moved about to cause the i~
gas-liberating reagent to flow into the sample compartment and to insure complete mixing of the reactants in the vessel. For that '. ~ _ ~_ ., ~ d ap ~

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: , - , . -~r~ 14 purpose, the base is equipped with an e~tension which serves as a convenient handle for manipulation of the apparatus.
Other advantages and objects of this invention will become apparent from the speciEication and drawings.
Drawings Figure 1 i9 a side elevational view of a micro-gasometer embodying the invention, the apparatus being illustrated in opened condition at the commencement of a sample-testing procedure.
~igure 2 is a side elevational vlew similar to Figure 1 but showing the apparatus ln closed conditlon ln a subsequent ~- step oE the operative procedure.
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Figure 3 is an enlaryed vertical sectional view ; showing the relationship between the reaction vessel and the remainder of the apparatus after the resilient gasket has been compressed -to adjust the meniscus of the indicating liquid of the manometer.

Figure 4 is a side elevational view illustrating a further step in the operation of the apparatus.

Figure 4a is an enlaryed vertical sectional view . of the reaction vessel illustrating somewl~at schematically 10 the intermixing of t.he reactants when the apparatus is ; tipped in the manner depicted in Figure 4.

, Figure 5 is a sicle elevational vi.ew illustrating . :
the condition of the apparatus upon the completion of the m .
gas-liberating reaction. ~ : .-.' ' .
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Referring to the drawings, the numeral 10 ;~; :
generally designates a.micro-gasometer embodying this -invent.ion and comprising a base 11, a manometer head assembly 12, a reaction vessel 13, and adjustable clamping means 14. ~ ~.
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Base 11 is rigid, paddle shaped, and provided . :
20 with a relatively narrow handle portion lla which is dimensioned to fit comfortably within a user's hand . :
(Figure 4). While it should be understood that the `~
dirnensions might be varied considerably, effective results ~.

have been achieved with a base having maximum width and length dimensions of about 2.0 and 6.6 inches, respectively, ;
and including a handle portion with width and length dimensions of about.l.3 and 2.4 lnches, respectively.

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The upper surface of the base's main body porti.on llb is provided with a well or depression 15 for receiving and locating the lower end of the removable and d:isposable reaction vessel 13. Spaced from the well, on opposite si~es thereof, are a pair of upstanding pivot blocks 16 and 17 which may be formed integrally with the base or, as in the illustration given, be formed as separate parts secured rigidly to the base by screws 18 or other fastening elements (Figure 3).

The manometer head assembly 12 is pivotally secured to upstanding block~l6 and may be shifted between the open or releasing position of Figure 1 and the lowered or vessel-engaging position of Figures 2 and 3. The head assembly comprises a generally rectangular plate member 19 having ~; tongue portions l9a and l9b at opposite ends thereof and :~ having a transverse slot 20 which extends from one side to the other and which receives a resilient contact member or gasket 21. ~s shown, the gasket is exposed from the underside of the plate~member through an enlarged opening 22 which has dimensions substan-tially greater than the top dimensions of vessel 13 (Figure 3).
: ' The manometer head assembly 12 also includes capillary tube 23 and capillary tube support 24. The support is rigidly fixed to plate member 19 and projects ~ -l upwardly when the plate member is in its lowered or closed position (Figure 2). At its extreme upper or distal end, . the support is provided with a transversely-projecting arm 2~a which is apertured at 25 (Figure 2) to receive the ~;
. upper end of the capillary tube 23. The lower end of the 30 tube extends through bore 26 in plate member 19 and through .~ opening 27 in gasket 21. To achieve a fluid-tight seal : . -l between the outer surface of the capillary tube and plate 19, ,' .

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the pla~e is counterbored at 28 to receive sealing ring 29.
The sealing ring fi-ts snugly about the outer surface of the capillary tube and sealingly engages the plate member 19 when the parts are assembled as illustrated in Figure 3.
The capillary manometer 23, which may take the form of a standard glass laboratory capillary tube of, for example, 100 microliter capacity, is therefore supported at'its open upper andlower ends, the upper end being received in ; opening 25 and the lower end being held frictionally in . ;
10 place by sealing ring 29 which is in turn ~ocked against axial displacement~(and sealingly engaged) by gasket 21.
1 It will be observed that the capillary tube locks the gasket against axial movement and that removal of the gasket first requires the capillary tube to be lifted (with a force sufficient to overcome the frictional resistance of ring 29) until the extreme lower end of the tube is disposed above the gasket. It will also be noted that the capillary tube . .:
is graduated, the graduation scale preferably being imprinted or otherwise applied to the tube support 24. The scale is :
20 conveniently graduated in millimeters although any other ~, appropriate measurement system may be utilized.

Tongue portion l9a of plate 19 is pivotally .
connected by transverse pin 30 to pivot block 16. For that purpose, the pivot block 16 may be formed in the shape of a clevis, having a pair of upstanding arms 16a which define ,. . .
:~ a space therebetween for receiving tongue portion ~9a.

Clamp 14 also takes the shape of a clevis, having ~ a transverse connecting portion 14a and a pair of depending ~ side portions 14b which straddle pivot block 17 and which ~ 30 axe pivotally connected to the block by transverse pin 31.

.; The transverse connecting portion l~a is provided with a threaded opening 32 which receives the threaded shank 33a of : .
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a knob-equipped adjustment member 33. As shown rnost clearly in Figures 2 and 3, the dlmensions of the space between the side arms 14b oE the clamp, and between the connecting portion 14a of the clamp and pivot blocl~ 17, are larger than the corresponding dimensions of the -tongue portion l9b at the free end of plate member 19~ The difference in the vertical dimensions is particularly significant because it permits limited pivotal (essentially verti~al) adjustment of the plate member 19, by means of rotating the knob-10 equipped adjustment member 33, after a realction vessel 13has been clamped ih position between pase 11 and manometer head 12. -The reaction vessel is generally rectangular in side elevation and in horizontal section, although other configurations might be acceptable. It is important, however, that the vessel be open topped, having an upper ; ` !~
rim 34 providing an upper surface 34a lying along a single plane. The interior of the vessel is divided into two compartments 35 and 36 by a transverse partition or septum 20 37. It will be noted that the partition extends transversely relative to the elongated base 11 and that it is substantially lower than the side walls 38 of the reaction vessel. The bottom surface 39 of the vessel lies along a plane parallel with the one defined by top surface 34a; therefore, the vessel may be securely and sealingly clamped between the ' manometer head and base in the manner illustrated.`

; The capillary tube of the manometer head contains a small quantity of any suitable liquid 40 that would be visible through the transparent wall of the tube. Any -30 non toxic aqueous dye solution may be used, the solution i preferably containing a surfactant (such as Triton X-100, marketed by Rohm & Haas Company, Philadelphia, Pennsylvania) to insure free or unrestrained movement of the dye solution ' ,. ,' . ' ;:~''`','`.,.'~ ' ' ' :
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within the capillary passage. While non-aqueous liquids might ' be used, they should have a specific gravity not appreciably greater than water to avoid or reduce the possibility that such liquids might escape ~rom the open-ended tube.
. In carrying out the method of the invention, a : reaction vessel 13, having one of its compar-tments 35 nearly . filled with a gas-liberating reagent R, is located in well 15 ; of base 11 as illustrated in Figure 1. The reagent may be an acid such as lactic acid (22 percent) as disclosed in patent 10 3,756,782. The amount of reagent in compartment 35 should be as close as possible to the capacity of that compartment with-out risking unintentional spilling of. the reagent into adjacent : compartment 36 during normal handling of the vessel and the ::
apparatus as a whole. Thus, if the capacity of compartment 35 (measured to the top of septum 37) is 0.6 milliliters, it has been found that such a compartment may conveniently receive 0.5 milliliters of reagent.
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; A measured quantity of biological fluid (bl-ood, -~
serum, or plasma) is pipetted into adjacent compartment 36 20 (Figure 1). The amount depends partly on the volume of reagent R in the adjacent compartment, it being essential . that the reag`ent be present in substantial excess. There-~ore, if compartment 35 contains 0.5 ml reagent (lactic acid), lOO~microliters (0.1 milliliters) of sample S may be placed on compartment 36.

.1, The manometer head 12 is then shifted into its closed position, and clamping means 14 is manipulated, to seal the open top of the reaction vessel so that it communicates only with the lower end of the capillary tube 30 23. Attention is directed to the fact that the body of indicator liquid 40 in the capillary tube will assume a position at the tube's extreme lower end when the tube is supported ~ertically (the surfactant contributing in : :~

`~` ' ' preventing the liquid from sticking in an elevated position), but that as resilient gasket or cushion 21 is compressed following initial sealing contact between the gasket and the vessel, li~uid ~0 is ~orced upwardly within the capillary tube because of the reduction in the total volume of the vessel's interior (Figure 3). Therefore, in operation of the apparatus, knob 33 of the clamping means is simply rotated to compress gasket 21 ùntil a meniscus o~ liquid 40 (pre~erably the upper meniscus) has reached a selected 10 starting point ~zero) on the scale. I

A modi~led operating procedure is to tighten the clamping means until the meniscus is slightly above the desired starting level (zero) and then reverse rotation of the knob, thereby relieving the compressi~e force, until khe meniscus has dropped to the zero point. Such a technique may be of value in minimizing drift in the operation of !~ .
the manometer resulting from creep or flow of the material o~ the resilient gasket.
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With the meniscus stabilized at the zero level, 20 the operator simply lifts the entire apparatus, gripping it ... .
by handle lla,' and tips it so that the gas liberating reagent ~lows from compartment 35 into compartment 36 (Figures 4 and 4a). Since the reagent is present in substantial excess, compartment 35 need not be emptied to :
produce complete reaction. Since tipping o the apparatus may be most easily accomplished by raising the clamp equipped end of the base (or lowering the handle end), it is apparent that in positioning the reaction vessel on the base in the initial step (Figure 1) the compartment 35 which contains 30 the reagent R should be located a greater distance from the handle than the sample-receiving compartment 36. Still holding the apparatus by its handle lla, the operator ~,-, : .- - . ,, . ~
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repetitiously moves the apparatus about any and/or all of : its axes to agitate the reactants. Such agitation should continue in a uniform manner for a measured interval such as, for example, thirty seconds. Thereafter, the level of the meniscus of liquid 40 is simply read from the scale of the manometer head.

The procedure is repeated with a standard solution of known carbon dioxide content substituted for the sample of biological fluid, and the trlue carbon 10 dioxide content of the sample is then calculated by the formula set forth in patent 3,756,782.

While in the foregoing, an embodiment of the invention has been disclosed in considerable detail for purposes of illustration, it will be understood by those skilled in the art that many oE these details may be ,~ varied without departing from the spirit and scope of '! the invention.

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Claims (16)

The Claims

1. A method of measuring the carbon dioxide content of a biological fluid, comprising the steps of introducing a measured sample of biological fluid into one portion of an open-topped reaction vessel containing in another portion thereof a reagent capable of liberating the carbon dioxide content of said biological fluid upon admixture therewith, then sealing said vessel so that the interior thereof communicates only with the lower end of a graduated transparent capillary tube containing an indicator liquid defining a meniscus visible through the wall of said tube, then adjusting the volume of the interior of said vessel to shift said indicator liquid in said tube until said meniscus is at a selected position along said graduated capillary tube, and thereafter mixing the contents of said reaction vessel while the same communicates only with said capillary tube to measure the amount of carbon dioxide liberated by observing the extent of displacement of said indicator liquid in said tube.

2. The method of Claim 1 in which said mixing step is performed by tipping said vessel and the capillary tube sealed thereto.

3. The method of Claim 2 in which said sample and reagent are supported in separate adjacent compartments of said reaction vessel, said mixing step including the tipping of said vessel so that at least a substantial portion of said reagent flows from its compartment into the adjacent sample-containing compartment.

4. The method of Claim 1 in which said sealing step includes securing a closure member upon the open top of said vessel with a resilient gasket interposed there-between, said adjusting step comprising the incremental pressing of said gasket until the volume of said vessel is reduced sufficiently to raise the meniscus of said indicator liquid to said selected position along said tube.

5. The method of Claim 4 in which said adjusting step includes preliminary compressing said gasket until said meniscus is displaced to a level slightly above said selected position, and thereafter reducing the compression of said gasket to lower the level of said meniscus to said selected position.

6. A device for measuring the carbon dioxide content of a biological fluid comprising a base adapted to support a reaction vessel, an open-topped reaction vessel removably supported upon said base, a manometer head mounted upon said base for movement between a raised position spaced from said reaction vessel and a lowered position in sealing engagement with the open top thereof, said head including a transparent capillary manometer tube, said tube communicating at its lower end with the interior of said reaction vessel when said head is lowered and containing an indicator liquid normally disposed at the tube's lower end, and clamping means for releasably securing said head in sealing engagement with said vessel and for altering slightly the position of said head when lowered to alter the volume of said vessel and thereby adjust the position of said liquid in said tube to a selected reference point.

7. The device of Claim 6 in which said head includes a resilient gasket engagable with the open top of said reaction vessel when said head is lowered, said gasket being compressible upon manipulation of said clamping means to reduce the sealed volume of said vessel and thereby alter the position of the indicator liquid within said capillary tube.

8. The device of Claim 6 in which said manometer head includes a plate member having an opening extending therethrough, said capillary tube having its lower end removably received in said opening of said plate member, and resilient sealing means engaging both said plate member and said capillary tube to form an air tight friction seal therebetween.

9. The device of Claim 6 in which said open-topped reaction vessel is provided with a generally vertical partition dividing the interior of said vessel into two adjacent compartments, said partition having an upper edge spaced below the open top of said vessel.

10. The device of Claim 6 in which said base is provided with an elongated handle extension dimensioned to be receivable within the hand of an operator.

11. The device of Claim 8 in which said clamping means includes a screw member equipped with a knob and engagable with said plate member for urging said plate member into sealing engagement with said vessel.

12. The device of Claim 11 in which said plate member is hingedly connected to said base, said plate member having a resilient gasket along the undersurface thereof for engaging the open top of said vessel, said gasket being compressible upon adjustment of said screw member to reduce the volume of the interior of the vessel when sealed and thereby adjust the position of the indicator liquid in said tube to a selected reference point.

13. The device of Claim 6 in which said base includes a well registrable with said manometer head when the same is in its lowered position, said well receiving the lower end of said reaction vessel.

14. The device of Claim 9 in which said vessel has a base wall and in which the open top of said vessel is defined by a surface extending along a plane substantially parallel with said base wall.

15. The device of Claim 14 in which said vessel has generally rectangular side walls.

16. The device of Claim 14 in which said vessel is formed of transparent material.