DEVICE AND PROCESS FOR ANALYSING GAS EFFECTS IN SAMPLES

Abstract

The invention provides a device and a process for analysing the effect of a concentration of a gas on a sample, especially measurement of the gas concentration within a sample, and/or measurement of the effect of the absorption and/or desorption of a gas by the sample, using spectrophotometric detection of a gas-sensitive and optically detectable analyte of the sample while measuring the concentration of the gas in a gas composition that is continuously delivered to each sample well in order to contact the samples with a gas having a predetermined and/or measured concentration, respectively a pre-determined and/or measured partial pressure.

Claims

1. A device for analyzing the effect of a gas in a sample, the device comprising: a housing enclosing at least one sample well, a gas conduit connected to the at least one sample well configured to permit gas to enter and to exit the at least one sample well, a gas source connected to an inlet of the gas conduit, a concentration device that is configured to determine the concentration of at least one component of the gas, and a spectrophotometer arranged to optically measure the at least one sample well.

2. The device according to claim 1, wherein the concentration device comprises a first gas sensor arranged at the inlet and a second gas sensor arranged at an outlet of the gas conduit.

3. The device according to claim 2, wherein both the first gas sensor and the second gas sensor comprise optical sensors containing a dye, that changes its emission in dependence on gas concentration.

4. The device according to claim 1, wherein the concentration device comprises or consists of a gas source configured to provide a pre-determined concentration of the at least one gas component, and wherein the at least one sample well comprises an array of at least 2 sample wells which are connected in series to the inlet.

5. The device according to claim 1, wherein the gas source is configured to deliver a gas flow having an increasing and/or a decreasing concentration of the at least one component of the gas over time or a gas flow having a constant concentration of all gas components of the at least one component of the gas over time.

6. The device according to claim 1, comprising a first pressure sensor configured to record gas pressure at the inlet, and a second pressure sensor configured to record gas pressure at an outlet of the gas conduit.

7. The device according to claim 1, comprising a conditioning device configured to temperature-control gas between the gas source and conduit to have a temperature at or above a temperature of the housing and configured to control humidity of the gas to saturation.

8. The device according to claim 1, wherein the at least one sample well comprises at least two sample wells connected in series to the inlet and an outlet of the gas conduit.

9. The device according to claim 3, wherein the at least one sample well comprises a dye which changes its emission in dependence on gas concentration, which dye is an oxygen-sensitive dye, a pH sensitive dye, and/or a carbon dioxide-sensitive dye, and the spectrophotometer is configured to optically measure the first gas sensor and the second gas sensor.

10. The device according to claim 1, wherein the at least one sample well comprises an array of sample wells, the housing comprises a bottom element in which the array and a lid element affixable to the bottom element configured to close the sample wells, and/or wherein the housing contains at least two separate gas conduits, each gas conduit connected to a separate array of sample wells and each gas conduit provided with a first gas sensor arranged at the inlet of the gas conduit and with a second gas sensor arranged at the outlet of the gas conduit.

11. The device according to claim 10, wherein the gas conduit comprises a groove in a region of the bottom element spaced from the bottom of the wells, which groove is circumferentially closable by the lid element.

12. The device according to claim 10, wherein the gas conduit is formed as a groove in the lid element, which groove can be interrupted in areas of the lid element covering wells.

13. The device according to claim 12, wherein each gas conduit connecting a separate array of sample wells is connected to a gas source configured to provide a different pre-determined gas composition.

14. The device according to claim 1, wherein the spectrophotometer is a microtiter plate reader, the device comprising a closure element adapted to close an opening of measuring chamber of the microtiter plate reader subsequent to arranging the housing inside the measuring chamber, which closure element accommodates at least a gas feed line connected to the inlet.

15. The device according to claim 1, wherein the spectrophotometer is connected to a computer configured to analyze light emanating from sample wells at at least one wavelength or at two or more different wavelengths, and to determine absorption for the at least one illumination wavelength, and wherein the computer is configured to determine local oxygen partial pressure of gas in each sample well, local PO.sub.2, by linear interpolation in accordance with
local PO.sub.2=inlet-PO.sub.2+((position of sample well along gas conduit/total number of sample wells along gas conduit)(inlet-PO.sub.2outlet-PO.sub.2)).

16. A process for analyzing the effect of a gas comprising depositing the at least one sample into one of an array of at least two sample wells contained in a housing, continuously delivering a gas composition containing a gas to the array of sample wells through a gas conduit in connection with each sample well, and spectrophotometrically concurrently measuring each sample well containing an aliquot of samples while in contact with gas.

17. The process according to claim 16, comprising spectrophotometrically detecting a gas-sensitive and optically detectable analyte of the at least one sample deposited in the at least one sample well at an inlet and outlet of a gas conduit connected to the array of sample well, measuring concentration of a gas by spectrophotometrically measuring signals of a first gas sensor and of a second gas sensor, which each are optical sensors containing an oxygen-sensitive dye, and determining the concentration of the gas in each sample well by interpolating between the measurements of the first gas sensor and of the second gas sensor.

18. The process according to claim 16, wherein the at least one sample is a whole blood sample, deposited as a droplet on a flat bottom surface of one of the at least two sample wells and is spread into a thin film by moving a stamp front surface against the bottom surface of the one of the at least two sample wells, and determining absorption and/or desorption of gas by the whole blood sample while at least one component of the gas composition has an increasing and/or a decreasing concentration, stepwise or continuous, over time during and/or between measurements.

19. The process according to claim 16, wherein aliquots of the at least one sample are deposited in sample wells of at least two separate arrays, wherein each gas conduit connecting the sample wells of one array is connected to a gas source that delivers a different gas composition, wherein a different constant gas composition is concurrently delivered to each array of sample wells, spectroscopically measuring each sample well, and interpolating between the measurement results obtained for aliquots of one sample.

20. A process for producing a device for analyzing the effect of a gas in a sample, comprising providing a microtiter plate reader as a spectrophotometer, arranging a housing comprising an array of sample wells connected in series by a gas conduit with its inlet connected to a gas source, the housing comprising an oxygen-sensitive dye arranged in a well of the housing as a first gas sensor, which well is arranged between the inlet and sample wells, and comprising an oxygen-sensitive dye arranged in a well of the housing as a second gas sensor, which well is arranged between sample wells and an outlet of the gas conduit, in a measuring chamber of the microtiter plate reader, and arranging a closure element for closing an opening of a measuring chamber, which closure element accommodates at least a gas feed line connected to the inlet of the gas conduit.

Description

[0051] The invention is now described in greater detail by way of examples and with reference to the figures, which show in

[0052] FIG. 1a a schematic overview of a device according to the invention,

[0053] FIG. 1b a schematic of an embodiment with multiple gas conduits of a housing for use in the invention,

[0054] FIG. 2a a housing for use in the invention, FIG. 2b shows an enlarged partial cross-section of a part of the housing of FIG. 2a, FIG. 2c shows a micrograph of a blood sample spread out in a sample well,

[0055] FIG. 3 shows measured (fluorescence lifetime of an oxygen-sensitive dye forming an oxygen sensor in the device of the invention) vs. calculated PO.sub.2,

[0056] FIG. 4 shows measured linear oxygen concentrations in sample wells connected by a gas conduit in series in the device of the invention,

[0057] FIG. 5 shows absorption spectra for whole blood under different gas flow, and

[0058] FIG. 6 shows gas absorption measurements of a blood sample in one sample well under a decreasing oxygen partial pressure in nitrogen,

[0059] FIG. 7 shows a mean of several oxygen dissociation curves (continuous line) and standard deviation (vertical error bars)

[0060] FIG. 8 shows an exemplary device, and

[0061] FIG. 9 shows a representation of measurement results obtained for one sample using two pre-determined gas compositions.

[0062] FIG. 1a shows a gas source 1 having two reservoirs 2 for gas components and a mixing valve 3 which is connected by a gas line 4 to a conditioning device 5 that temperature-controls the gas to a temperature of the temperature of the housing 6 or to a temperature above, e.g. 1 or 2 C. above the temperature of the housing 6. From the conditioning device 5, the gas line 4 is connected to an inlet 7 of a gas conduit 8 arranged within the housing 6, the gas conduit 8 leaving the housing 6 at outlet 9. The housing 6 contains 96 wells in a 812 array of wells, 92 of which are sample wells 10, a first gas sensor 11 is arranged in one well between the inlet 7 and the sample wells 10, and a second gas sensor 12 is arranged in one well between the sample wells 10 and the outlet 9. The first gas sensor 11 and the second gas sensor 12 consist of the oxygen-sensitive dye arranged on the bottom of their well. The gas conduit 8 from the first gas sensor 11 meanders along all wells 10 to the second gas sensor 12, connecting all sample wells 10 in series between the first gas sensor 11 and the second gas sensor 12.

[0063] The housing 6 encloses the openings of the wells 10 so that the gas entering the inlet 7 flows along the first gas sensor 11, along all of the sample wells 10, and along the second gas sensor 12. The housing 6 is arranged in the measuring chamber of a plate reader that forms the spectrophotometer 13. An entry opening of the spectrophotometer 13 is light-proof closed by a closure element 14, through which the gas line 4 is arranged for connection with the inlet 7 and a gas line 4 for connection of the outlet 9 to a suction pump 15. For measuring the pressure drop along the gas conduit 8, a first pressure sensor 16 is arranged in the gas line 4 leading to the inlet 7, and a second pressure sensor 17 is arranged in the gas line 4 between the outlet 9 and the suction pump 15.

[0064] FIG. 1b shows an embodiment of one housing 6 that contains 4 separate gas conduits 8, each with an individual inlet 7 for gas with a first gas sensor 11 arranged at the inlet 7, and an individual outlet 9 for gas with a second gas sensor 12 arranged at the outlet 9.

[0065] FIG. 2a in greater detail shows a housing 6 in top view with the array of the sample wells 10, a first gas sensor 11 between the inlet 7 and the sample wells 10, and a second gas sensor 12 between the sample wells 10 and the outlet 9. The gas conduit 8 that connects the sample wells 10 is arranged adjacent the plane of the top openings of wells 10 in the microtiter plate that forms a bottom element 6b with a lid element 6a in the form of an air-tight plastic cover of the housing 6.

[0066] FIG. 2b shows an enlarged cross-section of part of the housing 6 (wells H5, G5 and F5) with a bottom element 6b containing sample wells 10, a well (well H5) containing the first gas sensor 11 adjacent to the inlet 7, and the gas conduit 8 formed as a groove 8g in the upper portion and sealed by a lid element 6a. The gas conduit 8 does not directly connect well H5 to well G5 but via intermediate wells. In this embodiment, the gas conduit 8 is arranged in the bottom element 6b and opposite from the bottom 10b of sample wells 10. The direction of the gas flow is schematically indicated by arrows. As indicated by Abs., measurement can be absorption measurement through a sample S arranged on the bottom 10b of wells, and measurement of oxygen sensors 11, 12 can be by excitation irradiation Ex. and detection of emission Em..

[0067] FIG. 2c shows a micrograph of a blood sample spread out on the bottom 10b. This shows that a sample volume of 15 l can be spread out to a stable thin layer by moving a metal stamp against the sample that was pipetted onto the bottom 10b.

[0068] FIG. 3 depicts measurement of PO.sub.2 fluorescence lifetime of the oxygen-sensitive dye 200000023 SP-PSt3-NAU-D5-YOP, available from PreSens, Regensburg, Germany, in a plate reader used as the spectrophotometer in relation to calculated values, showing a highly linear correlation (Concordance correlation coefficient 0.999).

[0069] FIG. 4 shows the results of oxygen concentration measurements as partial pressure (PO.sub.2) with first and second oxygen sensors 11, 12 and an additional third oxygen sensor that was arranged in a sample well (well G12 in the microtiter plate element of the housing of FIG. 2a) under a gas having decreasing oxygen concentration, in relation to oxygen partial pressures calculated. This shows that also for a gradient of a gas component in the gas flowing along the gas conduit 8 through all the sample wells 10 in series, the gas concentration, exemplified by the oxygen partial pressure, can be interpolated from the signals of the first gas sensor 11 and the second gas sensor 12.

Example: Measurement of Oxygen Absorption by Whole Blood

[0070] In a device as shown in FIG. 2a, as a representative of a sample containing an oxygen sensitive analyte, whole blood was deposited into a sample well and spread out on the bottom of the sample well using a stamp. As a gas, a gas mixture containing an oxygen concentration of %, a carbon dioxide concentration of 5% and 75% nitrogen, for measuring the oxygenated state (HbO.sub.2) of haemoglobin was used and after flushing with nitrogen and carbon dioxide (5%), nitrogen and carbon dioxide (5%) was used for measuring the deoxygenated state (Hb). The gas was introduced into the gas conduit 8 of the housing 6. Measurements of first and second oxygen sensors 11, 12 were by irradiating with excitation wavelength of 543 nm and detecting emission at 653 nm every 1 min in a plate reader (Tecan) forming the spectrophotometer. Sample wells were illuminated at 415 nm and, separately at 431 nm, recording absorption every 1 min. Saturation (SO.sub.2) was calculated using the absorption ratio A.sub.431nm/A.sub.415nm., which ratio is independent of the sample volume.

[0071] The haemoglobin absorption spectrum of FIG. 5 shows typical curves, indicating that the device and process of the invention are suitable for such measurements.

[0072] Using a mixing device, the gas had an oxygen concentration decreasing from an initial 20% to 0% in nitrogen over 20 to 25 min. Measurements were made on a whole blood sample spread out on the flat bottom of one sample well (e.g. sample well position A6 of FIG. 2a) of a housing according to FIG. 2a. In addition to measuring the oxygen concentration by a first oxygen sensor and a second oxygen sensor, the pH was measured using a pH-dependent dye in an adjacent sample well covered with a sample aliquot. The housing was temperature-controlled to 37 C. in the measuring chamber of the plate reader. After 15 min, a plasma pH drift in the blood film by 0.05 pH units was detected. The pH drift is considered to be within a physiological range. This small pH drift that occurs after 15 min shows that the process according to the invention can be performed without buffer added to the sample, especially when determining the p50 and/or the ODC within 15 min of the analysing process. FIG. 6 shows the results (.box-tangle-solidup.=absorption at 431 nm, .diamond-solid.=absorption at 415 nm, .square-solid.=ratio of absorption at 431 nm/absorption at 415 nm, .circle-solid.=oxygen saturation), and also the p50 of the oxygen dissociation curve (ODC) that is determined at 15 min.

[0073] Effects of storage of blood samples at 0 to 5 C. was evaluated via repeated blood gas analysis of aliquots. Within 8 h of storage, plasma pH, COHb and MetHb did not change, whereas potassium levels and lactate slightly increased, and glucose and the anion gap slightly decreased. This shows that the process can reliably be performed using blood samples that were stored at 0 to 5 C. for up to 8 h.

[0074] When testing the intra-assay variability, it was found that within one housing comprising a microtiter plate (94 sample wells), the p50 values of the ODCs of whole blood are scattered with a mean Standard Deviation (SD) of 1.2 and a mean Coefficient of Variation (CV) of 0.04. This shows a fairly high precision and low intra-assay variability for the analytical process of the invention. This intra-assay variability, however, was found to strongly depend on data definition: it further strongly decreases with decreasing sample size, i.e. by pre-averaging the results from three (SD=0.72), six (SD=0.57). FIG. 7 shows the intra-assay variability is with the arithmetic means of several points (94 sample wells). It can be seen that SD is considerably higher in the steep part of the slope, levelling off in the flat, asymptotic ranges. Currently, it is therefore preferred to initiate the analytical process at oxygen concentrations (PO.sub.2) of at least 40 or at least 60 mm Hg, preferably at least 70 or at least 80 mm Hg.

[0075] It was found that the device and method of the invention provided for a very low inter-assay variability of measurement results when the process was performed by different laboratory workers. Currently, this very low inter-assay variability, and respectively a high reproducibility of the process is believed to be based on the temperature control of the housing, the temperature control of the gas delivered into the inlet of the housing, and the measurement of the gas concentration by the first gas sensor and the second gas sensor and measurement of the analyte with no or only a short delay, and on the set up of the device and the use of internal standard solutions.

[0076] A comparison of oxygen absorption measurement using standard haemoglobin solution (Equil QC 463) with a Hemox Analyzer (TCS Scientific Corp., USA) using a 100-fold dilution of the standard haemoglobin in accordance with the manufacturer's instruction for 118 sample measurements, each measurement lasting 30 min, gave a mean of 26.41.33 mm Hg, and using the device according to the invention using 10-fold diluted standard haemoglobin solution, in 3 runs, each for 94 sample wells in parallel containing triplicates of each sample lasting 30 min, gave a mean of 24.600.72 mm Hg (SD). In the set-up of the device according to the invention, a 100-fold dilution due to the thin film of the small sample volume gave no signal, and therefore a higher concentration of the standard haemoglobin solution was used. This also shows that the invention has the advantage of allowing the use of an undiluted, i.e. original, whole blood sample to be analysed, avoiding an influence of added reagents, e.g. avoiding dilution effects on the sample.

[0077] As a further comparison, blood samples of a female patient diagnosed with severe polycythaemia (Hb=18 g/dl) and a suspected high oxygen affinity hemoglobinopathy (p50<23 mm Hg) were tested. Aliquots of the blood samples were simultaneously analysed with the Hemox Analyzer. Importantly, p50 determinations were performed synchronously (24 h after venipuncture). A second blood sample from a healthy control person was analysed according to Mayo Clinic guidelines. Using the device according to the invention, a total of 120 ODCs from patient's blood and 72 ODCs from the control person were measured. When defining a result to be the mean of 3 sample wells, 40 independent p50 determinations were made. In the patient, p50 was 27.21.13 mmHg (meanSD) and thus slightly elevated (right-shift of ODC) compared with the healthy control (25.80.88 mm Hg).

[0078] Analyses of blood sample aliquots with blood gas analyzer, which is generally known to give an estimate of p50 with low accuracy only, the p50 of the patient was slightly increased over the healthy control, whereas both results were in the normal range.

[0079] In the Hemox Analyzer only one result was provided: p50=28.7 mmHg in the patient and p50=26.3 mmHg in the healthy control. This is the third independent and standard method to exclude a high oxygen affinity haemoglobinopathy in this patient. Further, this comparison shows that the device and process of the invention provide an accurate result in a shorter time.

[0080] FIG. 8 shows three separate arrays of sample wells contained in one housing, each array connected by a separate gas conduit that is connectable to a gas source. The array of sample wells 10a contains a first gas sensor 11 in one well that is arranged at the inlet 7 of the gas conduit 8 and a second gas sensor 12 in one well that is arranged at the outlet 9 of the gas conduit 8. The inlet 7 of the array of sample wells 10a is preferably connected to a gas source 1 that is set up to deliver at least two different gas compositions, preferably a continuously changing gas composition, e.g. a changing oxygen concentration (O.sub.2 ramp). The inlet of the gas conduit connecting the array of sample wells 10aa, and the inlet of the gas conduit connecting the array of sample wells 10aaa are each connected to a gas source that is set up to deliver, preferably to concurrently deliver, different pre-determined gas compositions having a stable composition. For example, array of sample wells 10aa can be connected to a gas source providing 3% (low O.sub.2) oxygen in water-saturated nitrogen, while parallel array of sample wells 10aaa can be connected to a gas source providing 5% oxygen (high O.sub.2) in water-saturated nitrogen. Samples to be tested, e.g. blood samples, are preferably provided as aliquots in corresponding sample wells, e.g. aliquots of one sample are deposited in sample wells having the same distance from the inlet 7 of the gas conduit 8 of each array of sample wells 10aa, 10aaa.

[0081] FIG. 9 for one sample shows measurements obtained in sample wells of parallel arrays of sample wells 10aa, 10aaa, each of the gas conduits that connect one of the arrays provided with a gas composition having a different oxygen concentration, expressed as oxygen partial pressure (PO.sub.2 (kPa)). These two measurements () are linearly interpolated or fit into a saturation curve, e.g. into a pre-determined sigmoidal Hill plot for oxygen saturation of hemoglobin, to determine the P50 value. This example shows that measurement of the analyte, herein hemoglobin, in two aliquots of one sample while in contact with different gas compositions can be sufficient for determining the saturation, e.g. the P50 value.

TABLE-US-00001 Reference numerals: 1 gas source 2 reservoir for gas component 3 mixing valve 4 gas feed line 5 conditioning device 6 housing 6a lid element 6b bottom element 7 inlet 8 gas conduit 8g groove 9 outlet 10, 10a, 10aa, 10aaa sample well 10b bottom of sample well 11 first gas sensor 12 second gas sensor 13 spectrophotometer 14 closure element 15 suction pump 16 first pressure sensor 17 second pressure sensor S sample