A METHOD OF PHOTOMETRIC IN VITRO DETERMINATION OF THE CONTENT OF AN ANALYTE IN A SAMPLE OF WHOLE BLOOD

Abstract: The method is a method of photometric in vitro determination of the content of an analyte in a sample of whole blood. The blood sample is transferred from an in vivo locality directly to the sample container of a sampling device. The sample container comprises a measuring chamber wherein there is provided a transparent body the radiation transmission characteristics of which vary with the content of analyte in the environment of the body. The transparent body is equilibrated with the sample and thereafter the measuring chamber is deformed in a controlled manner while the portion of the measuring chamber located in the radiation transmission path is drained for blood sample. The analyte content is determined on the basis of detection of the radiation transmitted from the radiation source through the deformed measuring chamber and the transparent body and to the radiation detector. A sampling device and an analyzer for use in photometric in vitro determination of the content of an analyte in a blood sample is also described.

Description: A METHOD OF PHOTOMETRIC IN VITRO DETERMINATION OF THE CONTENT OF AN ANALYTE IN A SAMPLE OF WHOLE BLOOD The present invention relates to a method of photometric in vitro determination of the content of an analyte in a sample of whole blood as set forth in the introductory part of claim 1. A number of clinical chemistry analyzers exists which analyzers consist of a combination of disposable compo¬ nents, which are only used for one single analysis ope¬ ration and only get in touch with one single sample, and an analysing section adapted for receiving the sample containing disposable device and containing the additio- nal components necessary for accomplishing a clinical chemical analysis. These analyzers all have in common that the transfer of blood sample from patient to disposable device takes pla- ce via a separate sampling device such as a syringe, a capillary tube or a vacuum tube sampling device. In certain cases the sampling device and the disposable device constitute one and the same device. Thus, Inter- national patent application WO 86/00138 (Shanks et al.) discloses a device with a cavity sufficiently small for a given sample to be sucked into the cavity by capillary effect. In this equipment the cavity is provided with an elec¬ trode structure and possibly a coating of a material adapted to the analysis to be performed with the equip¬ ment. The electrode structure provided in the cavity may i.a. be a potentiometric ion sensitive electrode struc- ture or an amperometric electrode structure. The latter is described in connection with determination of hydro¬ gen peroxide and oxygen in the sample. Supplementary use of the equipment for optical analysis of the products of a specific binding reaction is also described. From the specification of Danish patent publication DK 150804 (Lilja, «T.E. and Nilsson, S.E.L.) is known a sample container for sampling, mixing a sample with at least one reagent and directly performing a separate op¬ tical analysis of the sample mixed with the reagent. The sample container has a capillary cavity coated with a reagent and the inlet to the sample container works by capillary effect. The sample container is stated to be useful for most different kinds of analysis and to be especially advantageous for determination of hemoglobin. From the specification of British patent application GB 2025065 (Meiattini, F. et. al.) is known a plunger syringe for withdrawal of a blood sample. The blood sample is analysed by means of sensors incorporated in the syringe plunger. It is thereby avoided to transfer the sample to a sample station. The sensors are adapted for connection with an analyzer via conductors for regi- εtrating, processing, and outprinting analysis data. The specific sensors described in the specification of the said British patent application GB 2025065 are electro¬ chemical sensors for blood gases and blood electrolytes. None of the above-mentioned publications which in appli¬ cant's opinion represent the prior art closest to the present invention deal with the special problems con¬ nected with photometric determination of an analyte in whole blood. A serious problem when performing a photometric analysis of a particular analyte in whole blood is that the light reduction caused by the remaining components in the sample is extensive as compared to the light reduction attributable to the analyte. Another serious problem is that due to its content of blood cells whole blood gives rise to a light reduction depending on the content of blood cells in the sample, i.e. of the sample hematocrit, and accordingly said light reduction will constitute an unknown factor. Varying content of other components than hematocrit may also result in a light reduction which varies between samples. The object of the invention is to provide a method by which the above-mentioned problems connected with photo¬ metric analysis of a whole blood sample located in the sample container of a sampling device during the analysis procedure, are readily eliminated. This is achieved by the method according to the invention which method is characterized by the characteristic fea¬ tures of claim 1. "In vivo locality" denotes in the present context a lo- cality being in direct connection with the blood circu¬ lation or being a locality in the blood circulation it¬ self. Sampling by arterial puncture, whereby the blood sample is transferred from the artery to the sample con¬ tainer by a thin needle, as well as via an arterial ca- theter or via capillary puncture are sampling methodo¬ logies, in which the blood sample is transferred direct¬ ly from an dj vivo locality to a sample container. The radiation emitted from the radiation source may be broad banded as well as monochromatic radiation and may belong to the ultraviolet range, the visible range, the near infrared range and/or the infrared range. The radia¬ tion source may comprise one component or several compo¬ nents emitting radiation at their respective particular wavelength range. The radiation detector as well may comprise one component detecting radiation at a particu¬ lar wavelength range or several components detecting radiation at their respective particular wavelength ranges. The several components of the radiation source may be provided as one integrated device or as separate devices. The same applies to the radiation detector. The controlled deformation of the sample chamber is a de¬ formation reducing the volume of the measuring chamber to such extent that the measuring chamber is either com- pletely or locally drained for blood sample. By "local draining" is understood that the part of the measuring chamber traversed by the radiation is drained for blood sample. In a preferred embodiment of the method according to the invention the optical communication is established by placing the sample container in a sample container sta¬ tion of an analyzer. Alternatively, the optical communication may be establis¬ hed by one or several cables which via means for contact¬ ing the sample container and optical fibres establish op¬ tical communication between the optical system and the sample container. The transparent body used in the method according to the present invention is made of a material with a homegene- ouε structure, in particular a homogeneous surface making the material suited" for use in photometric transmission analysis. Preferably, the body is shaped as a disc or plate with two planoparallel surfaces and a thickness of less than 1 mm, preferably 1-500 urn, in particular 5-250 um and most preferably 8-200 um. If an indicator is fixed to the transparent body the in- dicator may be fixed by any suitable immobilization me¬ thod such as by absorption, adsorption, ion binding or covalent binding. Conceivably, it will be possible to make a determination of the content of several analytes in the blood sample within one measuring chamber provided that each indivi¬ dual analyte has an effect on the transmission properties of the transparent body and that each analyte affects the transmission properties differently. Such determination of several analytes within one measu¬ ring chamber may be accomplished by using a transparent body to which several indicators are fixed. The use of a transparent body containing a reagent which on reaction with the analyte forms a reaction product changing the transmission properties of the body in rela¬ tion to the analyte concentration in the environment of the body would be suitable for the determination of cer- tain analytes. In that case it may be the reaction pro¬ duct per se which changes the transmission properties of the body or an indicator which is fixed to the body and is affected by the reaction product. In particular, the method according to the invention aims at such analytes as blood gases, cationic and anionic components in blood (blood electrolytes), metabolites, hormones, enzymes, proteins, drugs and other chemical species which is or may be found in blood. Examples of _ 4 - such analytes are oxygen, carbon dioxide, H (or pH) , Li + , Na + , K + , Ca ++ , Mg ++ , Cl~, HC0 ~ , urea, glucose, creatinine, lactate, bilirubin, cholesterol, triglycerides, and uric acid. Beyond this the invention particularly aims at any analyte mentioned in Mosbach, K. , "Methods in Enzymology, Immobilized Enzymes and Cells, Part D", 137 (1988) p. 4-7 and ibid., p. 411. In connection with determination of the carbon dioxide content is preferred a body made of silicone rubber or another transparent, homogeneous material with a high dissolution power for CO_. The C0 2 determination is accomplished on the basis of testing the transmission properties of the body for 4.2 um radiation. The basic principles for such determination are disclosed in the specification of British patent application GB 2160646 and in the specification of European patent application EP 253559. In connection with determination of pH is preferred a bo¬ dy made of a polymer material with -OH groups available for immobilization, e.g. a cellulose or cellophane mate¬ rial, and with a pH indicator which may be covalently bound thereto, e.g. an indicator comprising the reactive group -S0 2 -CH 2 -CH 2 -0-S0 3 H. Cellophane in particu¬ lar has physical properties suitable for the present pur- pose, such as a well-defined thickness and a homogeneous structure. The invention also relates to a sampling device compris¬ ing a sample container with a measuring chamber having at least locally transparent opposite wall parts and having the characteristic features set forth in the characteriz¬ ing part of claim 11. Preferably, the locally transparent wall parts comprise planoparallel parts extending parallel with the planopa- rallel surfaces of the transparent body. By such configu- ration is ensured that the drainage of the measuring chamber is readily obtained, viz. by pressing the wall parts against the transparent body. The transparent body may be loosely accomodated in the measuring chamber or it may be fixed to a wall part. Other preferred embodiments of the sampling device ac¬ cording to the invention have the characteristic features set forth in the dependent claims. The sample container may comprise one measuring chamber or several measuring chambers disposed in series or in parallel. Preferably the sample container is made of an injection mouldable polymeric base material, e.g. the ma¬ TM tteerriiaall ssoolldd uunnddeerr the trade name ARNITE from AKZO, Arnhei , Holland. In connection with the performance of a pH or a carbon dioxide determination on a blood sample in the measuring chamber the wall parts of the measuring chamber are pre¬ ferably made of a polymer material having a low carbon dioxide permeability and suitable transmission properties for radiation at the wavelengths relevant to the above- mentioned determinations, said wavelengths being discus- sed at another page. A suitable material is an ethylene vviinnyyllaallccoohhooll ccooppoollyymeerr ooff the type EVAL-E TM sold by Kuraray Co., Osaka, Japan. In the case where the blood sample is provided by capil- lary puncture, the use of a sample container with a di¬ mension sufficiently small for the sample container to be filled by capillary effect is preferred. In the case where a sample of arterial blood is desired, the use of a sample container with an inlet located in a coupling means, preferably a Luer cone, for coupling the sample container to a needle or a catheter is preferred. The invention further comprises an analyzer for use in the method according to the invention, said analyzer having the characteristic features set forth in the characterizing part of claim 21. In a preferred embodiment the analyzer comprises data processing means for processing the registered radiation data for deriving the content of the actual analyte in the actual sample from these. Alternatively the analyzer is adapted for connection to a separate data processing unit. In a further preferred embodiment of the system accord¬ ing to the invention the analyzer comprises means for displaying the analyte content. Alternatively, the ana¬ lyzer is adapted for connection with means such as, e.g. a data screen, a display, a printer, or a plotter. The invention will now be explained in the following with reference to the drawings and the subsequent examples. In the drawings Fig. 1 is a perspective view of a preferred embodiment of an analyzer and a sample container, which together con¬ stitute the system according to the invention for photo¬ metric iτ__ vitro determination of an analyte in a blood sampler Fig. 2 is an enlarged schematic view from above of the sample container station of the analyzer with the sampl¬ ing device; Fig. 3, Fig. 4 and Fig. 5 are views of a preferred embo¬ diment of a sampling device according to the invention; Fig. 6 is an electric block diagram of the analyzer shown in Fig, 1; Fig. 7 shows the photometric basis for determination of pH; Fig. 8 is a perspective view of a sample container for use in determining pH; Fig. 9 is a partial cross section of an optical unit in a system comprising an analyzer according to the invention and a sampling device according to the invention for pho¬ tometric determination of pH and with a schematic re¬ presentation of the components forming parts of the opti¬ cal unit; In the different figures like reference numerals design¬ ate like parts. The analysis system shown in Fig. 1 and generally design¬ ated 10 is a compact portable "stand-alone" system, which is suited for decentral use, i.e. use outside a regular laboratory environment, e.g. in an operating room or at an intensive ward. The analysis system 10 comprises a blood sampling device 2 for disposable use and used in connection with an analyzer 11. The sampling device 2 is more explicitly described in connection with the descrip¬ tion of Fig. 3-5 below. The sample container 2 and the analyzer 11 are adapted to interact in the way that the analyzer 11 has a sample container station 1 with an op¬ tical section 3 adapted for receiving the sampling device 2 or possibly the sample container of the sampling de¬ vice, so that the optical communication between the sampling device 2 and the optical components of the opti¬ cal unit 3, which is necessary for photometric analysis, is obtained. The sample container station 1 can be closed by a cover 8, which is closed after placing the sampling device 2 in the station. By closing the cover 8 different mechanisms are activated, e.g. a not shown clamping mechanism, which secures the sampling device 2 in the optical section 3 and at the same time thermostatically controls the sampl¬ ing device to a desired temperature, preferably app. 37°C. Closing the cover 8 further results in a signal being sent to the controlling unit of the analyzer and indi- eating the start of an analysis procedure. An operator can control the operation of the analyzer by means of a keyboard 5 and a display 6. The analyzer 11 preferably also comprises a printer, which can produce an outprint 7 of i.a. the analysis results obtained by the analyzer. After placing the sampling device 2 in the sample con¬ tainer station 1 and closing the cover 8 of this, the optical components comprising of radiation sources and radiation detectors are activated, whereupon the ana- lyzer 11 calculates one or several parameters on the basis of the signals from the radiation detectors. The result of the calculations is displayed on the display 6 and is printed on the paper outprint 7 by the printer. When the calculations are terminated and the results displayed and/or printed out the cover 8 is opened and the sampling device 2 is displaced from the sample con¬ tainer station 1 and disposed of. In a larger scale Fig. 2 shows a partially schematic εec- tion of the sample container station 1 viewed from above. As shown the optical section 3 comprises four optical units 30, 40, 50, and 60 adapted for determination of pH (unit 30), carbon dioxide (unit 40), oxygen (unit 50) and hemoglobin and oxygen saturation (unit 60) respectively. The sample container of the sampling device with four measuring chambers is placed in a slot 4 in the optical section 3. Even though the analyzer 11 is shown containing four optical units it can in principle contain an arbitrary number and/or an arbitrary combination of units beyond the unit utilized in the method according to the inven¬ tion. Fig. 3 shows a longitudinal section of the sampling de- vice 2 and four segments of this in a larger scale. The sampling device comprises a body 23, which at least in specified areas is made of a material transparent for the relevant radiation. The body 23 has a continuous conduit 22 locally extended for forming measuring chambers 300, 400, 500, and 600. During a course of measurement the actual blood sample fills the conduit 22 from its inlet aperture 21 to a hydrophobic filter 24 placed behind the measuring chambers. The section of the body 23 surround¬ ing the inlet aperture is provided with a Luer cone and is therefore suitable for being mounted with a needle 20 of the type normally used for blood sampling. The section 25 of the body 23 pointing away from the inlet aperture is adapted for coupling with a traditional plunger sy¬ ringe. Such a plunger syringe is used as aid at the sampling in certain situations, e.g. when the patient, whose blood gas parameters are to be determined, has a very low blood pressure. When the sampling device 2 is placed correctly in the sample container station 1, the measuring chambers 300, 400, 500, and 600 communicate optically with the optical units 30, 40, 50, and 60. The measuring chamber 300 op¬ tically communicating with the optical unit 30 is adap¬ ted for determination of pH in the blood sample and con¬ tains a cellophane membrane 316, to which is immobilized a pH absorbance indicator. When the indicator is in che¬ mical equilibrium with the blood sample, the ratio be¬ tween the acid form of the indicator and the basic form of the indicator reflects the pH value of the blood sample. The chemical and photometric basis for the pH de- termination appears from Fig. 7, 8, and 9 and of the de¬ scription of these. An embodiment of the optical unit 30 appears from fig. 9 and the description of this. Finally it appears from Fig. 3 that the sampling device 2 has an integral needle protecting means 26. In the embo¬ diment shown the needle protecting means 26 is a tubular jacket movable in the axial direction of the sample con¬ tainer between a first position, wherein the jacket ex¬ poses the point of the needle, and a second position, wherein the jacket surrounds the point of the needle. Fig. 3 shows the protecting jacket 26 in the first posi¬ tion, in which it is placed at the sampling moment. Fig. 4 shows a longitudinal section of the sampling de¬ vice. The section iε placed perpendicular to the sec¬ tion shown in Fig. 3. The sample container body 23 consists of two halves, of which only one is seen in Fig. 4. Fig. 5 shows finally the same section as Fig. 3 but in Fig. 5 the protecting jacket iε displaced to the second position, wherein it surroundε the point of the needle. The jacket 26 iε displaced to this second position im¬ mediately after the sampling. Fig. 6 shows the electrical block diagram for the ana¬ lyzer 11 and speaks for itself. Fig. 7 shows the absorption spectrum for a pH absorption indicator N-9 more closely described below in connection with Fig. 8. The indicator is immobilized to a cello¬ phane membrane by the method also described in detail in connection with Fig. 8. The spectrum is recorded by a spectrophotometer of the type Shimadzu Spectrophotometer UV 250. The absorption measurements were performed on a cellophane membrane (6 x 12 x 0.028 mm) with immobilized indicator. The cellophane was placed in the cuvette of the spectrophotometer in a holder adapted thereto and with dimensions adapted to the dimensionε of the cuvette. In order to determine the abεorption conditions at dif¬ ferent pH-values, a number of pH buffers with pH values in the pH range from pH 6.14 - 11.74 was produced. Each buffer consisted of 500 ml 0.1 M KH 2 P0. to which was added the necessary amount of 0.1 M NaOH. The pH value of each buffer was potentio etrically measured (Radiometer PHM80) by means of a glass electrode (Radiometer GK2402C) , It appears from the spectrum shown in Fig. 7 that the acid form of the indicator has an absorption top at 458 nm and that the basic form of the indicator has an ab¬ εorption top at 595 nm and that the indicator not εub- εtantially absorbs radiation at wavelengths above 750 nm. Fig. 8 shows an embodiment of a sample container for use in determination of pH in a blood sample. The sample con¬ tainer generally designated 3000, is intended to interact with an analyzer with an optical unit as the one more cloεely deεcribed below in connection with Fig. 9. The εample container consists of two halves 3001 and 3002. These halves are made from a transparent plaεtic materi- al, e.g. softened polymethyl methacrylate of the type DE- GALAN TM SZ70 (Superfoε, Copenhagen, Denmark). The two halves are asεembled by pinε 3005 and 3006 in the half 3001 engaging corresponding, not εhown recesses in the half 3002, while not shown pins in this engage recesses 3007 and 3008 in the half 3001. The two halves are here¬ after welded together by ultrasonic welding. The line 3009 of material outlined on the lower half 3001 shown in the figure forms a welding seam after the welding. This line of material lies along the edge of a longitudinal conduit 3004, which centrally expands transverεely and for ε a meaεuring chamber 3011. A wall section 3010 of the upper half 3002 shown in the figure iε elaεtically deformable and for that reaεon haε a very reduced wall thicknesε compared to the rest of the half. Aε mentioned in connection with Fig. 7 a cellophane membrane 3003, to which there iε immobilized a pH indicator, iε placed in the measuring chamber 3011. Preparation of the Cellophane Membrane with Immobilized pH Indicator The pH indicator is delivered by Merck, Darmstadt, West Germany under the name N-9 and is known to contain the reactive group -SO2-CH2-CH2-O-SO3H. The immobilisation of the indicator to the membrane takes place in a process, whereby the above mentioned reactive group is transformed with sodium hydroxide into a vinyl sulphone group, which can be coupled to cello¬ phane previously activated by conditioning the cello¬ phane in 0.1 M NaOH for 15 minutes. The reaction scheme describing the coupling reaction be¬ tween the indicator and the cellophane membrane iε: R-SO2-CH2-CH2-O-SO3H + 2 NaOH (1) R-S0 -CH=CH2 + Na 2 S0 + 2 H 0 R-S0 2 -CH=CH 2 + HO-cellophane R-SO2-CH2-CH2-O-cellophane (2) R here designates an aryl group. The quantitative immobilisation procedure is the follow¬ ing: 100 mg cellophane (of the type 113650 - 36/32 from Struers, Copenhagen, Denmark) iε after the above men¬ tioned conditioning with NaOH prepared with an indicator solution consisting of 6 mg N-9, 1000 mg NaCl, 500 mg Na 2 C0_, 200 μl 8 M NaOH in 40 ml H 2 0. The cello¬ phane is left in the indicator solution for 30 minutes. Hereafter the cellophane is removed from the indicator εolution and waεhed several times with deionized water, whereafter it is left in deionized water at least through the night before use. The prepared membrane iε then kept in deionized water until uεe. Fig. 9 εhowε a prototype of an optical unit 30 for uεe in determination of pH in a blood sample. The blood sample is located in the measuring chamber 3011 in the sample container 3000, which is secured between a sta¬ tionary part 305 and a moving part 306. The previously mentioned membrane with immobilized pH-indicator is pro¬ vided in the measuring chamber. Before the measuring, the blood is pressed away from the measuring chamber by pressing the moving part 306 against the adjacent wall of the measuring chamber 3011. Preεsing out the blood sample from the measuring chamber results in the trans¬ mission conditions inside the measuring chamber not be¬ ing influenced by the blood sample itself. The optical unit 30 works in following way: A radiation source 301 in the form of a halogen lamp with a built-in lens of the type LNS-SE6-560 from Hamai Electric Lamp Co. TTD, Tokyo, Japan emits a parallel beam of broad- banded radiation. This radiation is transmitted to a heat absorbing filter 302 of the type KG 5 from Schott, Mainz, West Germany. This filter eliminates radiation from the infrared range. The radiation is transmitted from the filter to a depolarizer 303 and from there through the measuring chamber 3011. A silicon photodiode 304 of the type SFH 212 from Siemens, Munich, West Ger¬ many, receiveε radiation reflected from different sur¬ faces inside the optical unit and iε coupled to the ra¬ diation source 301 and ensures constant radiation inten- sity from thiε. After paεsage of the measuring chamber 3011, the radia¬ tion is focused by a lens 307 (ø 12 mm; f 12 mm; Ther- mooptik Arnold GmbH & Co., Weilburg, West Germany) onto three silicon photodiodes 310, 313 and 315, which are all of the type SFH 212 and situated in the focal plane of the lens 307. From the lens 307 the radiation is transmitted to a dichroic mirror 308 reflecting radia¬ tion of wavelengths lesε than 560 mm and transmitting radiation of longer wavelengths. The dichroic mirror is delivered by Optisk Laboratorium, Technical University of Denmark, Lyngby, Denmark. The εhort-waved part of the radiation paεses through a band-paεε filter 309 (centre value 458 nm; half band width 5.1 nm; Ferroperm, Ved- baek, Denmark) and iε transmitted from the band-pass filter 309 to a silicon photodiode 310 of the type SFH 212. The part of the radiation from the lens 307 trans¬ mitted through the dichroic mirror 308 iε tranεmitted to another dichroic mirror 311 reflecting radiation of wavelengthε leεε than 690 nm and tranεmitting the more long-waved radiation. The dichroic mirror is again de¬ livered by Optisk Laboratorium, Technical University of Denmark, Lyngby, Denmark. The reflected radiation is transmitted from the dichroic mirror 311 through a band¬ pass filter 312 (centre value 589 nm, half band width 14.8 nm; Ferroperm, Vedbaek, Denmark) and from there to a silicon photodiode 313 of the type SFH 212. The radia¬ tion transmitted through the dichroic mirror 311 is tranεmitted from here to a bandpaεs filter 314 (centre value 750 nm; half band width 10 nm; Ferroperm, Vedbaek, Denmark) and from there to a silicon photodiode 315 of the type SFH 212. The silicon photodiodes 310, 313 and 315 emit a current signal representing the intensity of 458 nm, 589 nm and 760 nm radiation, respectively. On the basis of these radiation intensities the pH value of the sample is calculated. More precisely the pH value of the sample is calculated in the following way: With different samples in the form of blood sample as well as the below mentioned two pH buffers in the sample container iε for each wavelength λ , λ , λ deter¬ mined the current I ϊac]ζσrounΛ •*. measured on the cor¬ responding photodiode for a sample container containing a cellophane membrane without immobilized indicator (i.e. a clear cellophane membrane) and sample. This cur¬ rent corresponds to I Q (cf. Lambert Beer's Law). From the knowledge of the dynamic range of the amplifier for the individual photodiode the current I b ac k groun d ,λ can be related to the abεorbance background t χ, as A background,λ = * lo 9 I background,λ where k is a constant. In the measuring situation where the sample container contains a cellophane membrane prepared with pH indica¬ tor, the current I m eaεurement,λ also relating to A measurement,λ * s determined, whereupon the "true" ab- εorbance at the wavelength λ is determined as A λ = -^measurement,λ ~ A background,λ' cf. A = log I - log IQ. To correct further for drift in the optical system, un¬ clear sample containers or other variations not directly relating to the pH determination, the absorbances A λl and Aλ,2„ are corrected with AA 1 3,' and the "new" ab- sorbance valueε are deεignated A 1 ,, and A 1 ,-. The relation to Lambert Beer' s Law hereafter appears as: A 'λl " A λl ~ A λ3 = A 'λ2 = A λ2 " λ3 = e X2,HIn *c HIn ,:L + ε λ2, In-' C ln -*1 where HIn designates the acid form of the pH indicator. In designates the basic form of the indicator, c τττ HIn and c τ n- the concentration of the acid and the basic form, respectively, of the indicator, ε the extinction coefficient for the wavelength and compound defined by the suffix, and 1 the pathlength of the radiation through the sample, which can be inverted to: C HIn = *11 A 'λl + k 12 'λ2 (4) In~ B *21 A 'λl + *22 A 'λ2 The constants k.., k 12 / ^2 1 ' and k 2 2 are deter ~ mined by measuring A λl » 2 and A λ 3 wit* 1 two P H buffers with known pH values as samples. For each of these pH buffers the proportion ci τ n„__./cx t i,m τ is deter- mined according to the Henderson Hasselbalch equation: pH - pk a + log[c In -/c HIn ] (5) Is it further supposed that c χ - + c H _ = 1, the two sets of connected values of c τ In- and c„Hl τ n can be calculated. If the corrected absorbance values are in- εerted in the equation set (4), four equations with the four unknown quantities -^I ' k 2 _i ' an ^ ^ 22 are obtained, and subsequently these four quantities can be calculated. By measuring unknown sampleε it iε hereafter poεεible to determine c H _ and c χ - from the equation set (4) and εubεequently pH from equation (5). With respect to the dimensionε of the εampling device iε noted that the measuring chamber preferably has a volume of 1 - 50 pi, more preferably 5 - 25 i an in particu¬ lar 7 - 12 pi and that the initial distance between the the transparent wall parts of the measuring chamber be¬ fore the drainage for blood sample is preferably less than 1 mm, in particular 200 - 700 pm. Further, is noted that after drainage a distance between the tranεparent wall partε in the region of the radiation tranεmission path is preferably 5 - 600 ^ m, in particular 8 - 300 um and most preferably 10 - 200 pm.

Claims: C L A I M S 1. A method of photometric dj vitro determination of the content of an analyte in a εample of whole blood wherein the sample from an in. vivo locality iε tranεferred directly to a εampling device com¬ prising a measuring chamber with at least locally transparent wall parts and the εample containing εample container is brought into optical communi¬ cation with an optical syεtem adapted to the ana¬ lyte and comprising a radiation source and a ra¬ diation detector arranged downstreams to the sample container in the radiation transmission path, c h a r a c t e r i z e d in, that there is provided within the measuring chamber a transparent body, the transmiεεion characteri- stics of which for radiation transmitted through the sample container vary with the content of ana- lyte in the environment of the body, that the body is equilibrated with the sample, that subsequently the meaεuring chamber iε deformed in a controlled manner thereby providing a substan¬ tially complete draining of blood sample from the part of the measuring chamber located in the radia¬ tion transmission path, and that the analyte content is determined on the basis of detection of radiation transmitted rom the ra¬ diation source through the deformed measuring chamber and the transparent body and to the radia¬ tion detector. 2. A method according to claim 1, c h a r a c t e r i z e d in, that there iε uεed a sampling device, wherein the transparent body has two planoparallel surfaces arranged perpendicular to the transmission direc¬ tion. 3. A method according to claim 1, c h a r a c t e r i z e d in, that there is used a sampling device, wherein the locally transparent wall parts of the measuring chamber comprise planoparallel parts extending parallel to the planoparallel εurfaceε of the transparent body. 4. A method according to claims 1 - 3, c h a r a c t e r i z e d in, that the analyte is a blood electrolyte, and that there is used a sampling device with a transparent body having fixed thereto an indicator, the radia¬ tion absorption characteristicε of which vary with the content of the particular analyte in the εample. 5. A method according to claim 4, c h a r a c t e r i z e d in, that the analyte is a cationic blood component, such as H + , Li + , Na + , K + , Ca ++ , Mg 6. A method according to claim 5, c h a r a c t e r i z e d in, that the analyte is H +, and that the indicator is a pH-indicator having a pronounced pH sensitivity at physiological blood pH values. 7. A method according to claim 6, c h a r a c t e r i z e d in, that the pH indicator is covalently bound to the transparent body. 8. A method according to claim 7, c h a r a c t e r i z e d in, that the transparent body iε provided from a poly¬ mer material compriεing -OH groupε available for immobilization, e.g. a cellulose or cellophane ma¬ terial. 9. A method according to claims 5 - 8, c h a r a c t e r i z e d in, that the pH indicator is a compound comprising the functional group -S0 2 -CH 2 -CH 2 -0-S0 3 H. 10. A method according to claimε 1 - 3, c h a r a c t e r i z e d in, that the analyte is C0 2 and that the transparent body is provided from a tranεparent material having a high dissolution power for C0 2 , e.g. εilicone rubber. 11. A sampling device compriεing a sample container with a measuring chamber with at least locally transparent opposite wall parts, c h a r a c t e r i z e d in, that there is provided within the measuring chamber a transparent body the radiation transmission cha¬ racteristicε of which vary with the content of a particular analyte in the environment of the body, and that the measuring chamber iε deformable in a con¬ trolled manner and such that it locally can be sub¬ stantially drained for sample. 12. A sampling device according to claim 11, c h a r a c t e r i z e d in, that the transparent body has two planoparallel surfaceε. 13. A sampling device according to claim 12, c h a r a c t e r i z e d in, that the locally transparent wall parts of the mea- suring chamber comprise planoparallel parts extend¬ ing parallel to the planoparallel surfaces of the transparent body. 14. A sampling device according to claims 11 - 13, c h a r a c t e r i z e d in, that one of the locally transparent wall parts is so deformable that it can be brought into abutment with the tranεparent body while expulsing any intermediate sample portion. 15. A εampling device according to claim 14, c h a r a c t e r i z e d in, that both locally tranεparent wall parts are so deformable that they can be brought into abutment with the transparent body while expulsing any intermediate sample portions. 16. A εampling device according to claim 14, 0 c h a r a c t e r i z e d in, that the locally transparent wall part is εo de¬ formable that it can be brought into abutment with the transparent wall part while expulsing any intermediate εample portion and that simultaneously 5 the transparent body iε brought into abutment with the oppoεite locally tranεparent wall part while expulsing any sample portion located between the body and the latter wall part. 0 17. A sampling device according to claims 14 - 16, c h a r a c t e r i z e d in, that to the transparent body there is fixed an in¬ dicator, the radiation absorption characteristics 25 of which vary with the content of the particular analyte. 18. A sampling device according to claims 11 - 17, 30. c h a r a c t e r i z e d in, that the transparent body is provided from a po¬ lymer material comprising -OH groups available for immobilization, e.g. a cellulose or cellophane ma¬ terial. 35 19. A sampling device according to claims 11 - 18, c h a r a c t e r i z e d in, that the indicator is a pH-indicator having a pro¬ nounced pH sensitivity at physiological blood pH valueε. 20. A sampling device according to claims 11 - 16, c h a r a c t e r i z e d in, that the transparent body iε provided from a transparent material having a high dissolution power for C0 2 , e.g. εilicone rubber. 21. An analyzer compriεing a radiation εouce, a radia¬ tion detector and a sample container station so arranged relatively to the radiation source and the radiation detector that a sample container arranged within the εample container station and comprising a meaεuring chamber with locally transparent wall parts is located within the radiation transmission path from the radiation source to the radiation de¬ tector, c h a r a c t e r i z e d in, that in the analyzer there iε provided meanε at the sample container station which meanε on activation are adapted to deform in a controlled manner the meaεuring chamber of a εample container arranged in the sample container station and that way that the measuring chamber part located in the radiation transmission path is esεentially drained for blood sample. 22. An analyεer according to claim 21 , c h a r a c t e r i z e d in, that the analyzer is adapted to cooperate with a εample container compriεing a tranεparent body, and that the meanε for controlled deformation of the measuring chamber cooperate with a wall part of the measuring chamber and displace and deform εaid wall part that way that the wall part iε brought into abutment with a surface of the tranεparent body and the oppoεite surface of the transparent body iε brought into abutment with the oppoεite wall part of the measuring chamber while any intermediate blood sample portions are expulsed from the radia- ion transmiεsion path.

Inventors: ANDERSEN WILLY, JENSEN NIELS-HENRIK, LUNDSGAARD FINN CHRESTEN, ANDERSEN, WILLY, JENSEN, NIELS-HENRIK, LUNDSGAARD, FINN, CHRESTEN
Applicants: RADIOMETER AS, RADIOMETER A/S
Application year: 1989
Main classification: G01N33
IPC codes: G01N21, G01N33, G01N35