GB2352511A - Colorimeter having an LED light source - Google Patents

Abstract

A portable colorimeter, eg for analysing the haemoglobin content of blood samples, comprises a housing having an LED light source (20), a light detector (50), a sample chamber (120), an output display, and a power supply eg battery and/or mains. The light beam (10) from the source passes via an interference filter (180) through a solution (30) in a cuvette (40) to the detector (20). The detector converts the detected light Intensity into a corresponding frequency of electrical pulses which are fed to microcontroller. A further detector (80) detects any variation in the source output, eg due to temperature changes, and adjusts the current supplied to the LED via a servo loop to keep the source intensity constant. The sample chamber is closed by a removable plug (130) and a hinged lid (170).

Description

2352511 COLOURIMETRY DEVICE - This invention relates to a colourimetry

device. In particular, a colourimetry device for analysing the haemoglobin content of blood samples.

Photometry is the technique of detecting light radiation and changes in radiation intensity. If the measured light is in the visible range of the electromagnetic spectrum, i.e. 200 to 380 nm, then the term applied to the analysis is colourimetry.

The basic components of a colourimeter are a light source, a wavelength selector (usually in the form of a number of filters), a sample container (usually a cuvette), a photodetector (to detect the light which is not absorbed by the sample), and a display (to indicate the result of the colourimetric analysis).

The technique involves a beam of light which passes through a cuvette containing the sample to be analysed. The sample is usually in the form of a solution. The light is provided by a white light source, and the beam of light is passed through a colour filter or alternative wavelength selection device. The incident light then passes through a cuvette containing a chemical compofind in solution. The intensity of the light leaving the sample will be less than the light entering the cuvette. The loss of light or absorption is proportional to the concentration of the compound. The extent to which the light is absorbed by a sample is dependant upon many factors. The main factors are the wavelength of the incident light and the colour of the solution. Each compound in solution has a typical (and usually unique) absorption spectrum, i.e. a unique pattern of the amount of absorption at each of the individual wavelengths in the spectrum. The absorption of the sample is compared with that of standards containing known amounts of the analyte thereby enabling the concentration in the sample of the analyte to be estimated.

In most cases the spectrum will have a peak, i.e. a wavelength at which absorption is at a maximum. This is often referred to as the X max.

2 If the absorption is being quantified, it is essential that it is measured as close as possible to the X max, as sensitivity is reduced at any other wavelength. Therefore, for greatest sensitivity and linearity, it is essential to limit the measuring wavelength to the area of highest absorption.

Colourimetric devices are well known in the art. However, these colourimetric devices are relatively expensive due to the nature of the components required to provide the accuracy of measurement which is needed in order to detect and to measure the light penetrating through the sample being measured. For example, the light beam is provided by a white light source, which in most cases is a tungsten lamp. In order to limit the measuring wavelength to the wavelength at which the compound to be detected has the highest sensitivity, the white light beam must be treated to produce a monochromatic wavelength which is the X max for the compound to be detected. There are several ways by which the white light beam may be limited to produce monochromatic radiation, e.g. gelatin filters, interference filters, or diffraction gratings, may be used. However, each of these filters has its own drawbacks.

Gelatin filters are low cost selection devices which produce or transmit a wide band of radiation, i.e. 20 mn. The most common type of filter is constructed by sandwiching a thin layer of dyed gelatin of the desired colour between two thin glass plates. However, there are two drawbacks to using gelatin filters:

1. They have a wide bandpass which can lead to non linearity in standard curves.

2. They absorb approximately 30 to 40 % of all incident radiation thereby reducing energy throughput to the detector.

Coloured glass filters may be used as selection devices in colourimeters, although they have very wide band passes, again leading to non linearity in standard curves. However, more specific wavelengths can be achieved using a combination of glass filters. However, not surprisingly, the more filters which are used, the more expensive and bulky the final colourimeter. To ensure that all wavelengths in the visible spectrum are catered for, 3 approximately eight gelatin filters are required. These filters are expensive and the presence of several filters within the one colouritneter increases the cost and the size of the colourimeter.

Interference filters are used to select wavelengths more accurately by providing a narrow bandpass of around 10 nm. This type of filter also only absorbs approximately 10 % of the incident radiation over the whole spectrum, thereby allowing light of higher intensity to reach the detector. However, as is the case with the gelatin filters, several interference filters are incorporated in prior art colourimeters in order to ensure that all wavelengths in the visible spectrum are provided for.

Finally, diffraction gratings may be used. These are generally fixed in position and the radiation exiting the diffraction grating is reflected by means of a rotatable mounted miffor. However, this requires that the position of the miffor be accurately calibrated whilst the colourimeter is in a stable stationary position, and once calibrated, the colourimeter should not be moved or bumped as this can alter the position of the miffor, thereby resulting in the use of a monochromatic radiation of an incorrect wavelength relative to the X max of the compound to be detected.

If, during use, chemicals or sample solutions are splashed or spilt into the euvette chamber of a prior art colourimeter, it can be a timeconsuming and complicated procedure to remove the chamber and to clean it out. It is possible that during this process the chamber may be dropped and damaged, or tiny parts of the assembly, such as clips or screws, may be mislaid, rendering the colourimeter either incapable of providing accurate measurements, or unable to be reassembled to make any further measurements.

Further, due to the number of components and the way in which they are held in position, colourimeters of the prior art are bulky bench top items of equipment which are sensitive to movement and are not readily transportable. Therefore, these colourimeters are not readily useable by travelling health care workers and the like particularly in the field in developing countries.

4 Apother problem with prior art colourimeters relates to the way in which the sample containers are held in the sample chambers of the colourimeter. It is important that the sample containers are not scratched, as this reduces the light transmission through the sample container, and affects the accuracy of the colourimetric reading. Often, disposable sample containers are used, and these sample containers are often scratched by the retaining clip provided in prior art colourimeters to hold the sample container in position in the sample chamber. This has the effect that such disposal plastic containers cannot be used more than once before disposal.

In addition, due to the use of white light sources such as tungsten lamps, prior art colourimeters are only practical for use in regions with stable mains power supplies. This, in combination with their non- portability, generally limits their use to laboratory premises in the western hemisphere, despite their potential use as a diagnostic tool for conditions such as anaemia in developing nations.

The colour intensity in the solution of the sample which is measured may be an inherent property of the solution, or it may be developed by the addition of suitable reagent(s) to the solution. The addition of a suitable reagent may result in a chemical reaction involving the compound of interest, which generates a detectable change in the absorption of the solution.

For example, the haemoglobin cyanide method of assaying the level of haemoglobin in a blood sample involves the addition of Drabkin's solution (a toxic cyanide-containing reagent) which converts haemoglobin derivatives to stable end products. It is the absorbance properties of the stable end products which are measured to determine the amount of haemoglobin which was originally in the blood sample.

However, as is the case with the haemoglobin cyanide assay, often the reagents used are extremely dangerous or unpleasant to work with. Therefore, it is desirable to develop alternative assays which do not require the use of dangerous reagents.

For example, the alkaline haernatin D-575 method of assaying the levels of haemoglobin in a blood sample involves dilution of the sample in a nontoxic alkaline solution (Alkaline haematin-D (AHD) reagent) which converts haemoglobin derivatives into a stable end product; alkaline haematin D-575 which has a X max at 575 mn.

Prior art colourimeters are capable of measuring the absorption of samples at any selected monochromatic wavelength within the visible spectrum. As mentioned above, this leads to an increase in the size and the cost of the devices. This capability to measure across all wavelengths of visible light is often not exploited by the users of the colourimeter, particularly where the sample measurements being carried out are routine and the machine is always set to detect compounds at a single wavelength. For example, a laboratory which is concerned only with analysing the levels of haemoglobin in blood samples, will consistently use the colourimeter at a particular wavelength, selected to be appropriate for the assay being used, despite the ability of the colourimeter to measure at all wavelengths across the visible spectrum.

It is an object of the present invention to address the problems of the prior art.

The inventors have developed a colourimeter device which in a preferred embodiment is transportable, capable of operating in conditions where there is an absence of mains power, avoids the expense of including multiple filters, and can be easily cleaned in the event of spillage of sample solutions or reagents Accordingly, a first aspect of the invention provides a colourimeter comprising a LED light source. The colourimeter may be portable to allow its convenient use in different locations.

A housing with a sample receiving means may be provided in which a sample may be received for colourimetric analysis.

The colourimeter may comprise a light detector arranged to detect light emitted by the LED light source after the light has passed through the sample.

6 The colourimetric measurement obtained may be displayed by an output display.

The LED light source may be a narrow bandwidth LED. For example, the LED light source may be an amber LED light source with a restricted wavelength range.

The LED light source may be a bright light monochrome LED. For example, the monochrome LED may have a peak wavelength of 574mn, and the half bandwidth (50% of peak emission) may be around I I mn.

If for example, an amber LED is used, a filter such, as an interference filter, may be placed in front of the LED to farther restrict the wavelength range of the light. If a monochrome LED is used, a filter may still be provided in front of the LED light source. However, this filter may be specific for blocking infrared (IR) radiation, which can be an undesirable wavelength which may be emitted using this LED light source type.

The colourimeter may operate using any one of a choice of power sources, such as mains voltage, an internal battery, or some other external power source such as a generator or external battery pack. Further, these power sources may be prioritised. For example, if mains voltage is available, the colourimeter will preferentially use it. However, if mains power is unavailable the colourimeter will preferably use power from any available external power source. In the event that neither mains voltage nor external power are available, the colourimeter will operate using its own rechargeable internal batteries. In addition, when other power sources are available, the internal batteries will automatically recharge.

Further, should mains power or an external power source fail during use of the colourimeter, the colourimeter will automatically switch to internal battery power without interruption. The colourimeter will then automatically switch back to mains voltage or external power source if they become available once again.

7 This makes the colorimeter capable of use in areas where mains power is either unavailable or unreliable, such as in developing countries or countries suffering from civil war or natural disaster.

The light detector may be a light intensity to frequency converter, thereby detecting the intensity of the light that is not absorbed by the sample solution, and producing a frequency output corresponding to the light intensity.

in addition, the colourimeter may comprise an intensity monitor. This monitors the intensity of the light issued by the light source after the light has passed through any filters present and before the light reaches the sample solution.

The intensity monitor detects variations in the intensity of light emitted by the LED light source. This variation in light intensity may be caused by the LED light source heating up overtime. The intensity monitor may be connected to a servo loop such that any variations in light intensity detected by the intensity monitor are fed back to the servo loop which alters the drive current of the LED light source accordingly, so as to increase or decrease the intensity of light emitted by the LED light source, and maintain the emitted light at a constant intensity.

The frequency output from the light detector may be input into a micro controller which converts the frequency to an output reading by comparison with calibrated values held in the microcontroller. These calibrated values are obtained at the beginning of each set of sample measurements by measuring the absorbance of a standard sample solution of known composition and concentration.

The sample receiving means of the colourimeter may comprise a through channel in the housing, and be provided with a removable end plug or cap. The sample may be held in the sample receiving means by a container such as a cuvette. The container may be held in position in relation to the light beam in the sample receiving means.

8 Preferably, the container is held in position by resilient biasing means which acts against the container. For example the resilient biasing means may be in the form of a clip.

Further, the through channel may also be provided with a hinged lid at the opposite end such that the through channel may be sealed from external light.

The through channel may comprise a removable collar which is inserted into the through channel and seals the interior of the channel from the LED such that, once the end plug has been removed, fluids may be flushed through the through channel for cleaning purposes.

The colourimeter may be used to measure the absorbance of any assay sample solutions, merely by providing the colourimeter with an LED light source of the appropriate wavelength range, and a filter of the appropriate wavelength range selection specificity.

For example, the colourimeter may be used to measure the absorbance of an assay sample solution to determine the haemoglobin level of blood.

Although it is possible to assay for the haemoglobin levels of an undiluted blood sample, if a diluted blood sample is used, the amount of blood required is very much lower. For example, an assay using diluted blood only requires around 30 [LI of blood.

In order to avoid the use of toxic reagents such as haemoglobin cyanide, a haemoglobin assay may be used wherein the blood sample is diluted in AHD lysing solution. AHD lysing solution is both non-hazardous, and easily obtainable.

A fin-ther aspect of the invention provides a method of colourimetric analysis comprising measuring the light absorbed by a sample positioned in a light beam, in which the light beam is generated by an LED light source.

Colourimeters in accordance with the present invention will now be described, by way of example only, with reference to accompanying Figures 1 to 5 in which; 9 Figure I shows a schematic diagram of some of the components of an embodiment of the invention; and Figure 2 shows a cross section through the sample chamber of the embodiment of Figure 1.

Figure 3 shows a circuit diagram of instrument display, keypad and microcontroller sections of the embodiment of Figure 1.

Figure 4 shows a circuit diagram of an LED intensity servo loop of the embodiment of Figure 1.

Figure 5 shows a circuit diagram of an instrument power supply of the embodiment of Figure 1.

Figure 1 shows a schematic diagram of an embodiment of a colourimeter according to the invention, where a light beam 10, is emitted by the LED light source 20. The light beam 10 then passes through the sample solution 30 in the sample cuvette 40, and any light not absorbed by the sample solution 30 is detected and quantified by a light detector 50. The light detector 50 is a light to frequency converter, and the intensity of the light detected is converted to a corresponding frequency of electrical pulses which are input into a PIC microcontroller 60 (PIC is a Registered Trade Mark of Microchip Technology Inc.) As the LED light source 20 heats up, the intensity and spectral characteristics of the light emitted by the LED light source 20 change. It is important that this change in intensity is either included in calculations to determine the absorbance of light by the sample solution 30, or else the intensity of the light emitted is controlled at a constant level. Otherwise, erroneous measurements of sample solution absorbance of light may be made.

Figure I shows a feedback mechanism 70 comprising an intensity detector 80, a servo loop 90, and a voltage reference 100. The intensity detector 80 detects any changes in the intensity of the light emitted by the LED light source 20, and this information is transmitted to the servo loop 90, which increases or decreases the current supplied to the LED light source 20 by a suitable amount to either increase or decrease the intensity of the light emitted by the LED light source 20, thereby maintaining the intensity of the light emitted at a constant level.

Figure 2 shows a cross-section through the colourimeter 110 so as to show a cross-sectional view of the sample chamber 120. The sample chamber 120 can be sealed from external light entering the sample chamber 120, by a removable end plug 130 at one end, and a hinged lid 170 at the opposite end. Although the end plug 130 may be a friction fit into the sample chamber, in the embodiment shown in Figure 2, the end plug 130 is held in place using a screw 140.

The end plug includes a resilient retaining clip 150 which extends from the end plug 130 into the sample chamber 120. The retaining clip 150 is oriented so as to avoid interrupting the path of the light-beam 10 emitted from the LED light source 20 whih passes through the sample solution 30 in the sample cuvette 40 before being detected and quantified by the light detector 50. In an alternative embodiment, the retaining clip 150 does not extend as high as the point in the sample chamber 120 where the light beam 10 passes through the sample solution 30.

The LED light source 20 and the intensity detector 80 are positioned in a second chamber 160 which intersects the sample chamber 120. The light detector 50 is positioned in the second chamber 160, on the opposite side of the sample chamber 120 to the LED light source 20 and the intensity detector 80.

in use, the sample cuvette 40, containing the sample solution 30 ready for colourimetric analysis, is placed into the sample chamber 120 where it is held in the correct orientation relative to the light beam 10 by the retaining clip 150.

The sample cuvette 40 is held by the retaining clip 150 such that the light beam 10 passes through the sample cuvette at an angle perpendicular to the plane of the sample cuvette 40. This ensures that each time a sample cuvette 40 is placed in the sample chamber 120, the sample cuvette 40 is held in the same orientation relative to the light beam 10, and the light 11 beam 10 passes through the same distance 'of sample solution 30 each time. If the distance of sample solution 3 0 through which the light beam 10 passes were to vary, then the greater the distance, the more light would be absorbed and the less accurate and repeatable the final measurement would be.

In order to avoid scratching of the sample cuvette 40, as this would reduce the light transmission through the sample cuvette 40 and affect the accuracy of the colourimetric reading, the sample chamber 120 has a circular cross section. Therefore, when the sample cuvette 40 is pressed against the inside of the sample chamber 120 it will only contact the sample cuvette 40 at the comers. In addition, the sample cuvette 40 will be held perpendicular to the plane of the light beam 10. By providing a retaining clip 150 which presses against the sample cuvette 40 in the same plane as the light beam 10, or at right angles to the plane of the light beam 10, one face of the sample cuvette 40 will always be normal to the light beam 10.

Once the sample cuvette 40 has been inserted into the sample chamber 120, the hinged lid 170 is closed, thereby sealing the chamber from external light.

At this point, the colourimeter may be activated to make a measurement, for example, by pressing a button or switch on the exterior of the colourimeter (not shown). Once the colourimeter has been activated, and before the LED light source 20 is switched on, the light detector 50 is activated. If there is any light in. the sample chamber 120, for example, due to the hinged lid 170 not be properly closed, the microcontroller will prevent the LED light source from switching on and will show a warning message on the display of the colourimeter (not shown) to alert an operator that the sample chamber is not sealed to external light. This prevents the colourimeter from making measurements which will be inaccurate due to external light influencing the measurement, as well as preventing wastage of the life of the LED light source 20 on such measurements.

Providing no external light is detected by the light detector 50, the LED light source 20 is activated, and a light beam 10 is emitted. This light beam 10 passes through an interference filter 180 positioned in front of the LED light source 20. The interference 12 filter 180 screens the wavelengths of light emitted by the LED light source 20, and restricts the wavelength to a narrower band range, for example, the wavelengths penetrating the interference filter 18 0 may be 5 8 Onm 1 Onm.

The narrow range wavelength light passing through the interference filter 180 travels through the sample solution 30 in the sample euvette 40, where, depending on the absorbance properties of the sample solution 30, a proportion of the light is absorbed by the sample solution 30. The remaining light which passes through the sample solution 30 is detected by the light detector 50, which converts the light intensity detected to a variable frequency pulse train, i.e. the greater the intensity of the light detected by the light detector 50, the greater the frequency of the pulse train.

This pulse train is input into a PIC microcontroller 190, which converts the pulse train frequency into a standard colourimeter output reading.

If, for example, the colourimeter is preprogrammed to operate at 580nm 10nm to measure the haemoglobin content of diluted blood samples, the PIC microcontroller will be programmed to convert the pulse train frequency to an output measurement of the haemoglobin level of the sample in g/dI.

Alkaline haematin D-575 method of measuring haemoglobin levels in blood samples 1. Alkaline haematin-D (AHD) reagent was prepared by dissolving 25.Og of Triton X-100 (Aldrich Chemical, Cat No. 23472-9) into 1 000ml of 0. 1 M NaOH.

2. A control standard (AHD std) was prepared by dissolving 36mg of chlorohaemin into IO.Oml of the AHD reagent. (This control standard when diluted 1:151 should read 0.26A in a colourimeter at wavelength of 575 nin, and is equivalent to a blood sample of 9.0g/dI.

3. The evaluation of the haemoglobin level was carried out as follows:

13 a. 100 whole blood samples, anticoagulated in EDTA, were obtained.

b. The blood samples were diluted in duplicate in AHD reagent (1: 15 1) by carefully pipetting 10tl of blood into 1.5ml of the AHD reagent. The blood was pipetted calibrated capillary tubes. The alkaline haematin control standard was similarly diluted 1: 15 1, by pipetting I Ogl of the standard into 1.5ml of the AHD reagent.

c. The standard and blood samples diluted in the AHD reagent were read against a reagent blank in a colourimeter, (peak transmission 580 nm) and results, which were converted by the colourimeter to haemoglobin concentrations in g/dl, were recorded.

The colourimeter is supplied with an LED type HLMT-CLOO light source (Hewlett Packard) or a LED type TLYH180P (Toshiba), with an interference filter such as a 580run interference filter 2-5801 (Optometrics (UK) Ltd). Alternatively, an LED type L200CWGIK (Ledtronics) or TLGA 183P (Toshiba) may be used, without an interference filter.

Claims (25)

14 Claims

1. A colourimeter comprising an LED light source.

2. A colourimeter according to claim I wherein the colourimeter is portable.

3. A colourimeter according to claim I or claim 2 comprising a housing with a sample receiving means in which a sample is received for colourimetric analysis.

4. A colourimeter according to claim 3 comprising a light detector arranged to detect light emitted by the LED light source after the light has passed through the sample.

5. A colourimeter according to any preceding claim comprising an output display to display theresults of the colourimetric analysis.

6. A colourinieter according to any preceding claim w herein the LED light source is a narrow bandwidth LED.

7. A colourimeter according to any preceding claim wherein the LED emits monochromatic light.

8. A colourimeter according to any preceding claim comprising a single wavelength selector filter.

9. A colourimeter according to claim 8 wherein the filter is a an interference filter.

10. A colourimeter according to any preceding claim wherein the colourimeter operates on mains voltage and/or an external energy source other than mains voltage.

11. A colourimeter according to claim 6 wherein the colourimeter can also operate using a battery as the energy source.

12. A colourimeter according to claim I I wherein the battery is rechargeable.

13. A colourimeter according to claim I I or claim 12 wherein the battery is an internal battery.

14. A colourimeter according to any preceding claim wherein the light detector is a light intensity to frequency converter.

15. A colourimeter according to any preceding claim comprising an intensity detector.

16. A colourimeter according to claim 15 wherein the intensity detector detects variation in the intensity of light emitted by the LED, and acts by means of a feedback loop to regulate the current drive to the LED, thereby maintaining the light emitted by the LED at a constant intensity.

17. A colourimeter according to any one of claims 14 to 16 wherein the frequency output from the light detector is input to a micro controller which converts the frequency to an output reading by comparison with calibrated values held in the micro controller.

18. A colourimeter according to any preceding claim wherein the sample receiving means comprises a through channel in the housing, with a removable end plug or cap.

19. A colourimeter according to claim 18 comprising a removable collar which is inserted into the through channel and seals the interior of the channel from the LED light source such that, once the end plug or cap has been removed, fluids may be flushed through the through channel for cleaning purposes.

20. An assay system using a colourimeter according to any preceding claim.

21. An assay system according to claim 20 wherein the assay system is used to determine the haernoglobin level of a blood sample.

16 22, An assay according to claim 21 wherein the blood samples are diluted.

23. An assay according to claim 22 wherein the blood samples are diluted in AHD lysing solution.

24. A method of carrying out an assay according to any one of claims 21 to 23 comprising:

1. Preparation of a blood sample diluted in AHD reagent lysing solution, in a sample container: and 2. Inserting the sample container into the sample receiving means of the colourimeter.

3. Activating the colourimeter to obtain a measurement of the haemoglobin level in the diluted blood sample, based on the light absorption properties of the diluted sample.

25. A method of colourimetric analysis comprising measuring the light absorbed by a sample positioned in a light beam, in which the light beam is generated by an LED light source.