WO2012038930A1

WO2012038930A1 - System of the "point of care" type for measuring total plasma bilirubin especially of neonates

Info

Publication number: WO2012038930A1
Application number: PCT/IB2011/054192
Authority: WO
Inventors: Richard Partridge Wennberg; Claudio Tiribelli; Giorgio Bergamo; Sandro Tognana; Antonio Maria Berto
Original assignee: Fondazione Italiana Fegato-Onlus; Microlab Elettronica S.A.S. Di Bergamo Giorgio & C.; Innovation Factory S.R.L.
Priority date: 2010-09-23
Filing date: 2011-09-23
Publication date: 2012-03-29
Also published as: ITMI20101730A1

Abstract

The present invention relates to a device and a method for determining the plasma bilirubin level especially in neonates. The system for measuring plasma bilirubin comprises an instrument having a reflectometer, able to detect the plasma bilirubin in a blood sample from which the respective corpuscular portion was removed on a membrane suitable for the purpose and a strip or stick comprising a filter and the membrane. The system consisting of the instrument and the strip can measure plasma bilirubin with an accuracy of +/- 5% at bilirubin concentrations in the range 3-30 mg/dL (approx. 50- 500 μΜοl/L) at haematocrit level up to 60% on blood samples of just 20 - 25 μl.

Description

SYSTEM OF THE "POINT OF CARE" TYPE FOR MEASURING TOTAL PLASMA

BILIRUBIN, ESPECIALLY OF NEONATES

***** ***** *****

Field of the invention

The invention relates to a system comprising a reading device having a reflectometer able to detect total plasma bilirubin from a plasma sample deprived of the respective corpuscular portion and a strip that is able to separate the plasma from a sample of whole blood. The invention further relates to the method of determination of plasma bilirubin concentration by means of said system and to the use of the system comprising the reading device and the strip.

Prior art

Transient jaundice due to unconjugated free bilirubin in the blood occurs in about 30% of neonates and is usually benign. A bilirubin level of 25 mg/dL is regarded as the maximum safe limit in term-born neonates. In fact, at high plasma levels, unconjugated bilirubin can cross_. the blood-brain barrier, causing serious, permanent brain damage (principally kernicterus). It is therefore important to monitor the bilirubin level in jaundiced neonates to determine those neonates requiring suitable treatment for lowering the plasma bilirubin levels. Current practice in the more developed countries is to monitor serum bilirubin levels before neonates are discharged. This test is performed routinely by clinical laboratories using methods known by a person skilled in the art based on diazo reactions that generate coloured complexes measurable spectrophotometrically and requires from 0.5 to 1.0 ml of blood. The bilirubin level can also be determined non-invasively by instrumental techniques, as no other yellow pigments are present in neonates. A sufficiently accurate measurement can therefore be obtained by measuring the optical density of the plasma at 450-460 nm, which is the wavelength of the absorption peak of bilirubin. An instrument (UB Analyzer, Arrows Inc; Osaka, Japan) measures both the total bilirubin and the free bilirubin, i.e. not bound to blood plasma proteins, but at present it is not available in the USA or Europe. Instead, a portable instrument is in use, consisting of a transcutaneous reflectometer (BiliCheck®, Respironics Inc., Murrysville, PA). The latter is an instrument offering the advantage that it is non-invasive and is sufficiently accurate for screening bilirubin levels up to about 15 mg/dL. However, it also has many disadvantages, especially for use in developing countries where kernicterus is still one of the major health problems, or in medical studies. The main drawbacks of this instrument of the "point of care" type (also designated POC hereinafter) currently in use are: 1 ) limited accuracy at high plasma bilirubin levels, 2) the need for calibration at each measurement, 3) the need for frequent battery recharging, 4) high costs both of acquisition and of use, having a selling price above€ 2500 and above€ 3-5 for calibration for each determination.

Alternative strategies are under development and are briefly described below.

A haemofluorometer of the "point of care" type is currently being developed for measuring bilirubin on whole blood. The relation between blood bilirubin and plasma bilirubin depends on the binding affinity and the concentration of red blood cells. Clinical levels of correlation between blood and serum bilirubin have not yet been established.

Microfluidic systems for measuring plasma bilirubin are also under development, but the techniques currently available for these systems allow plasma to be separated from blood with limitation to haematocrit values below 45%.

The main problem in developing POC systems based on the use of plasma is that of obtaining a sufficient amount of plasma in the presence of high haematocrit levels (50-60% or more), which are common in neonates.

This problem of treating neonates with jaundice does in fact occur in the advanced countries, if it is borne in mind that in the USA and in Europe about 4 million and 6.5 million neonates respectively are discharged from neonatal centres. The problem is even greater in developing countries, where hyperbilirubinaemia and kernicterus are among the major causes of long-term disability.

US02002/140940 relates to the analysis of blood samples in the very broad sense. With regard to bilirubin, this states that there are absorption peaks at certain wavelengths. In this connection, reference is made to a figure (3), which does not show the spectrum of bilirubin.

An optical probe is also described, whose reflectance is sent to a spectrometer, which separates and quantifies the various wavelengths.

This system has on the one hand the problem that a spectrometer must be available, and on the other hand the problem that the high levels of haemoglobin in whole blood, i.e. not separated into its components, obscure the contribution of reflectance from bilirubin, leading to unreliable results.

In the description, reference is made to a sample containing plasma and red blood cells (haematocrit). This passage probably refers to the separation of residual red blood cells and not to the case when the red blood cells constitute 50-70% of the blood, as in neonates. These can in fact only be separated by special filters, while Fig. 6 and the associated description show that the hydrophilic separating membrane is identical to the membrane where the reading is taken. The material of both is stated as being cellulose, nitrocellulose or PVDF.

JP5924765 shows a blood/plasma separating system. However, this system requires an enormous volume of blood to obtain a sufficient amount of plasma, which is transferred vertically onto a membrane for determination. This solution is not satisfactory in the case of neonates, from whom it is difficult to take a large volume of blood. Moreover, the vertical transfer of the plasma makes the device particularly thick and hence inconvenient both for storage and for use.

Summary of the invention

One aim of the present invention is therefore to satisfy these needs, by overcoming the limitations of the transcutaneous reflectometer (e.g., BiliCheck) currently in use, and of those in development.

The aim is thus to find a system for measuring total plasma bilirubin in the presence of high haematocrit levels with a high level of accuracy, that is easy to use and inexpensive.

Now, the inventors found that the aims of the invention are fulfilled by a system comprising a reading device (or reader) of low cost having a reflectometer and a strip, which requires just 20-25 μΙ of blood for executing measurement of total bilirubin and which has a reproducibility of 8% for amounts of total bilirubin greater than 30 mg/dL. The present invention relates to a method of measuring the total bilirubin present in the plasma separated from a blood sample in a predetermined amount, the method comprising the following steps:

- first irradiation of the plasma with blue light,

- first measurement of a respective reflected radiation (reflectance) according to said first irradiation,

- based on said first measurement, calculation of a bilirubin concentration by means of a linearized function approximating a bilirubin absorption curve as a function of its concentration according to said first irradiation.

Blue light in fact incorporates substantially the whole absorption spectrum of the reflectance of bilirubin. Thus, in contrast to the prior art, the plasma is not irradiated only with radiation corresponding to the absorption peak, nor is the reflectance separated into bands of different frequency.

According to a preferred variant of the method, it also comprises the following steps:

- second irradiation of the plasma with green light,

- second measurement of a respective reflected radiation (reflectance) according to said second irradiation,

- based on said second measurement, calculation of a value of contamination of the plasma with haemoglobin.

Green light corresponds substantially to the whole absorption spectrum of haemoglobin and not only to its absorption peak. Higher levels of reflectance according to green light indicate greater contamination of the plasma and therefore a lower reliability of said bilirubin measurement.

The plasma sample can in fact be contaminated as a result of haemolysis of the red blood cells with release of haemoglobin into said plasma, which is why the bilirubin measurement is unreliable.

On the contrary, by providing means for irradiation with green light it is possible to obtain an indication of the level of contamination of the plasma sample and of the reliability of the bilirubin measurement performed. The contamination with haemoglobin can be determined before or after bilirubin measurement.

Advantageously, the use of LEDs proves particularly economical relative to the known procedures that aim to irradiate exactly at the wavelength of the absorption peaks of the substances to be detected.

Moreover, the use of blue LEDs, i.e. measuring the whole absorption spectrum, makes the measurement less sensitive to the haematocrit levels.

The method of the present invention also comprises preliminary separation of the plasma from the corpuscular portion of the blood.

Another aim of the present invention is to supply a strip for separating the plasma from a residual corpuscular portion of a predefined amount of blood, in such a way that this amount is greatly reduced and in order to perform it successfully on blood of neonates, i.e. having percentages of red blood cells between 50 and 70% of total blood.

Therefore the present invention further relates to a device for separating the plasma from a residual corpuscular portion of a predefined amount of blood consisting of a strip for separating the plasma from the corpuscular portion of a blood sample formed from a membrane and a filter that are partially overlapped, included in a support according to claim 9.

Another aim of the present invention is to supply a reader of devices for separating the plasma from a residual corpuscular portion of a predefined amount of blood, able to perform measurements of total bilirubin, in particular on neonates, which proves to be particularly economical and reliable.

The present invention also relates to a strip reader for separating the plasma from a residual corpuscular portion of a predefined amount of blood according to claim 14. The system that has been developed for measuring total bilirubin will be described in detail below with reference to a preferred embodiment both of the reading device, or reflectometer, and of the disposable strips suitable for separating the plasma from a sample of whole blood taken from a subject whose bilirubinaemia is to be measured. The dependent claims describe preferred embodiments of the invention, forming an integral part of the present description.

Brief description of the drawings

The description of the reading device and of the strips for separating the plasma is given below with reference to the appended drawings, which are supplied for purposes of illustrating but not limiting the present invention.

Fig. 1 shows the variation of reflectance in relation to the total bilirubin concentration;

Figs. 2a and 2b show views, perpendicular to each other, of a first variant of strip;

Figs. 3a and 3b show views, perpendicular to each other, of a second variant of strip;

Fig. 3b is shown with parts removed;

Fig. 3c shows a semifinished version of the variant shown in Figs. 3a and 3b;

Fig. 4 shows a part of the disposable strip reading device;

Fig. 5 shows a logic diagram of a first variant of disposable strip reading device;

Figs. 6 shows a logic diagram of a second variant of disposable strip reading device;

Figs. 7 and 8 show flow charts of the operation of the devices according to Figs. 5 and 6.

The same reference numbers and letters in the figures identify the same elements or components.

Detailed description of the invention

The system for measuring plasma bilirubin is based on a method of measuring the total plasma bilirubin from a sample of whole blood comprising a step of depositing a sample of whole blood on a filter partially overlapping a membrane so that the filter holds back the corpuscular portion of the blood and the membrane receives the liquid portion (i.e. the plasma) without using any chemical reagents.

Thus, the method envisages measuring bilirubin by optical reflection by irradiating the membrane comprising the plasma with blue light radiation that contains substantially the whole absorption spectrum of bilirubin and the associated absorption peak that has a wavelength of about 470nm.

Since this wavelength represents an absorption peak for bilirubin and since the curve of reflection becomes almost linear on a negative logarithmic scale (see Fig. 1 ) as the bilirubin concentration increases, then the measured reflection represents a proportional linear measure of the total bilirubin contained in the blood according to a negative logarithmic scale.

This curve is obtained by irradiating the plasma of blood samples, containing known levels of bilirubin, with blue light. This is equivalent to calibrating the measuring instrument, i.e. the strip reader, taking into account not only the absorption peaks, but the whole absorption spectrum.

A first advantage of this method is that it does not require a spectrometer, and it is not necessary to know the parameter known as "molar extinction", which is required for the operation of many spectrometers.

Advantageously, this method requires a very small amount of whole blood, in fact a prototype measuring system, which is also an object of the present invention, was able to perform reliable measurements with just 20-25 μΙ of blood. Moreover, the complete absence of chemical reagents and extreme simplicity of use make the system easily portable and usable by anyone, mainly because no preliminary operation is required for separating the plasma from the corpuscular portion of the blood, which is separated automatically by the strip as conceived.

According to another aspect of the invention, the method of measurement is performed by a plasma bilirubin measuring system comprising, referring to Figs. 2 to 4, a reader 1 having a reflectometer (Figs. 5, 6) and a separating device, i.e. a strip comprising a support, of the liquid plasma portion from a residual corpuscular portion of a predefined amount of blood 2, for simplicity called strip hereinafter (Figs. 2, 3). The strip (or stick) 2 is described here with reference to Figs. 2a and 2b, and comprises a supporting band 21 housing a filter 22 and a membrane 23.

The purpose of the filter 22 is to separate the corpuscular portion of the blood from the plasma, which is transferred to a membrane 23 that absorbs it for the purpose of performing said optical measurement by reflection with the reader 1.

The supporting band 21 is in the form of a strip of material that is sufficiently rigid to be inserted in a reading opening of reader 1 .

A membrane 23 and a filter 22 partially overlapping the membrane 23, in a portion 24, are attached to the same face of the supporting band. Via the overlap 24 between membrane and filter, the membrane 23 absorbs the liquid component of the blood deposited on filter 22.

Advantageously, the membrane 23 and the filter 22 are, with the sole exception of the overlap 24, stretched on the support 21. Therefore the strip is substantially flat and simple both to manipulate and to store.

According to a preferred variant of the strip, it has a total length of about 3 centimetres and a width of about 3-7 mm, moreover the filter 22 has a length of 1 .5- 13 mm and the membrane 23 a length of 6 millimetres, with the overlap 24 of about 0.5-3 mm, comprising an overlap surface of 2-6 mm², while the width of the membrane and of the filter is about 4 mm, so the membrane has a surface area of between 20 and 30 mm² with an optimum of 24 mm².

The strip dimensions are not unimportant, bearing in mind that the time required for filtering the plasma and saturating the membrane increases with increase in haematocrit and with the filter dimensions. It is preferable for the membrane to be of nitrocellulose and to have characteristics similar to Prima™ 85, which has capillarity between 70 and 105 (s/4 cm) and calibre 200 (μιτι).

A filter for separating the plasma from the corpuscular portion of the blood can be selected from those known by a person skilled in the art and preferably made of a material consisting of a matrix of glass fibre and polyester that retains the blood cells and allows the plasma to pass through.

It was found that with a nitrocellulose membrane having a surface of 20-30 mm², or an optimum of 24 mm², and a filter having a surface of 40-55 mm², with an optimum of 48 mm², it is possible for a sample of about 20μΙ of neonate blood to be treated adequately. In fact, different surface areas require saturation times that are unsuitable for the type of measurement, even leading to false measurements in relation to the haematocrit concentration.

It is preferable, as shown in Figs. 2a and 2b, for the supporting band 21 to be slightly wider than the filter and the membrane, so as to have the lateral edges free of the supporting band and possibly of increased thickness, so that filter and membrane are contained within the outer surfaces of the strip. Figs. 3a, 3b and 3c show a preferred variant of strip 80.

This comprises a containing support 99 that integrates a supporting band 81 substantially identical or similar to the supporting band 21 , on which a membrane 83 and a filter 82 are arranged, partially mutually overlapping so that they define an overlap 84. This is similar to strip 2.

The containing support 99 comprises a groove 85 in which said band 81 is inserted. The same containing support 99 can define the supporting band 81.

As can be seen from Fig. 3c, this containing support 99 is obtained by folding a plastic or plastic-coated sheet, in the manner of origami.

The containing support 99 thus has a flat longitudinal shape, the groove extending parallel to this longitudinal form, preferably along the mid-line of the containing support 99.

Referring to Fig. 3a, a fin 86 is hinged to said containing support 99, with a rotation axis transverse to said longitudinal shape, on the face of the containing support 99 that comprises the groove 85 corresponding to the overlap 84 between membrane 83 and filter 82.

Advantageously, the fact that filter 82 and membrane 83 are housed in said groove 85 means that they are protected from the lateral edges of said groove, avoiding any contact with an internal part of the strip reader, which would lead to contamination thereof or soiling of its active parts.

This fin 86 is voluntarily not shown in Fig. 3b, for greater clarity of the figure.

This containing support can be made, advantageously, by folding sheets of paper, preferably plastic-coated, or plastic and similar materials.

The advantage of making the containing support 99 by folding a sheet of plastic- coated paper or a sheet of PVC and similar materials provides the undoubted advantage of not having to provide expensive moulds. This must also be evaluated in particular bearing in mind the presence of the fin 86 connected to the containing support 99 as described above.

In this way, when the fin is folded over on membrane 83, during deposition of the blood sample on filter 82, it cannot fall directly onto membrane 83, making the whole strip unusable.

Next, when the fin 86 is folded over on filter 82, this prevents direct contact of the operator's fingers with the filter. Accordingly, when the blood is deposited on filter 82, fin 86 is folded over on the latter, uncovering the membrane 83, which thus remains free for subsequent irradiation.

Moreover, by grasping the strip 80 and the associated fin 86 between the fingers, the fin can be dimensioned and connected to the containing support 99 to produce a slight pressure on filter 82. Advantageously, this pressure further facilitates passage of the plasma into the membrane.

Thus, this fin 86 defines a constricting and covering element of filter 82.

This advantage is even clearer when the thickness of the filter is greater than the thickness of the membrane, for which the operation of constriction is almost automatic as a result of folding over of the fin to cover the filter.

With particular reference to Fig. 3c, the semifinished article of the containing support 99 consists of a plastic-coated or plastic sheet punched to define a kind of cross with openings 97, made so that by folding the punch along the dotted edges, the filter and the membrane remain uncovered, always taking into account the position of the fin 86.

A particular object of the present invention is the strip 80 obtained by folding a plastic or plastic-coated sheet, as described here.

The reader 1 , described here with reference to Fig. 5, comprises processing means 1 1 and a reflectometer comprising a housing 10 which houses a photodiode 12 and, preferably, a first blue LED 13 with emission preferably centred on 470nm and a second green LED 18 centred on an emission wavelength of 570nm.

The photodiode 12 and the LEDs 13 and 18 are inserted in a housing 10 that has a groove 10' for receiving said strip 2.

Fig. 4 shows schematically an exploded view of the housing 10 (and 30). This comprises a first lower part 101 which houses two pins with a sprung ball 102 and 102' and in which the groove 10' is made. Moreover, the housing comprises an upper part 103, comprising an optical collector 104, intended to be attached to said first lower part 101 so that the collector is aligned with the membrane of the strip.

Above the collector, said LEDs 13 and 18 and photodiode 12 are connected to a printed circuit 105. The printed circuit is connected to the collector 104 for measuring the reflectance of the membrane.

The collector 104 can be made directly in the upper part 103 by milling and/or drilling. The membrane has a width of about 4 mm, therefore the hole for measurement, through which the optical radiation passes, must have a slightly smaller diameter, with an optimum value of 3.2 mm. This is so as to be able to ensure, with a certain tolerance, that the membrane receives and emits simultaneously the light coming from the LEDs and photodiode that are located immediately above said membrane. The collector 104, preferably metallic, comprises a through cavity, preferably countersunk, the reflective walls of which contribute to uniform distribution of the luminous intensity that impinges on the strip, compensating the misaligned position of the two LEDs relative to the photodiode and contributing to high repeatability of the measurements.

According to the solution depicted in Fig. 4, these three components 12, 13 and 18 are positioned perpendicularly to the longitudinal axis of the reader but this positioning is not obligatory.

The optical collector 104 collects the light from the membrane and sends it to the photodiode, which converts it into an electrical signal. This signal is suitably amplified in the optical reader 1 , by a first amplifier 15 and a second amplifier 15' or third amplifier 15" depending on which of the blue or green LEDs is in operation (Fig. 5). The amplified signal is then sent to an analogue/digital converter, preferably integrated in the microprocessor 1 1 , to be acquired and processed by the microprocessor.

The source of blue light is used for performing the bilirubin readings.

Preferably, the optional source of green light is used for evaluating any contamination of the plasma by the haemoglobin contained in the red blood cells. Green light is in fact preferentially absorbed by haemoglobin, which is red, which could interfere with the measurement of bilirubin. The microprocessor 11 filters, with a low-pass filter, the signal received from the amplification section (15,15', 15") to remove some of the noise that accompanies the signal. Filtering of the signal is preferably executed by the firmware of the microprocessor 11 so as not to slow down the response time of the amplification section 15,15', 15" and of the optional offset correcting circuit 14.

Another preferred variant can comprise a single amplification stage with programmable gain controlled by the microprocessor 1 1 , for example a digi-pot or a programmable operational amplifier, rather than the cascade of two amplifiers as shown in Figs. 5 and 6.

The software filter currently used is preferably of the FIR type with 256 taps; however, various implementations such as MR or a different number of taps are equally valid. Any hardware filtering must be fast enough to take account of the reading times.

The luminous intensity of the blue LEDs and optionally of the green LED is, moreover, compensated directly by the microprocessor 1 1 or by the optional compensating circuit 14 connected to an input of said first amplifier 15 and to said microprocessor 11 as a function of the variations in ambient temperature and the variations in voltage of the batteries, etc., the reader shown in the present variant being supplied by a suitable battery power supply 88.

The firmware, moreover, envisages displaying a bilirubin value, calculated according to the calibration curve stored in the microprocessor, and coinciding with that shown in Fig. 1 , on the display 16'.

For this purpose the reader is provided with an alphanumeric display 16' that shows the result and supplies the operator with the messages necessary for correct use and operation of the reader, with which he can interact via a keypad 16.

The data visualized on the display can be sent to a personal computer via a USB port controlled by the microprocessor 11.

In order to minimize the phenomenon of "bleaching" or of degradation of the bilirubin caused by the light, the LEDs are switched on for a very short time, preferably less than a second.

Moreover, according to a preferred embodiment of the invention, the reader comprises a microswitch or a further optical sensor (not shown) for detecting strip insertion.

Owing to said microswitch or optical sensor, the microprocessor 1 1 acquires the moment of strip insertion, from which a predefined time for stabilization of the measurements is counted. When said stabilization has not been achieved within a further predefined time interval, the reader indicates a time-out error.

This precaution ensures that diffusion of the plasma in the strip is optimum. When the strip is inserted in the reader, the reflected light is read every 5-10 s and it is considered that diffusion is complete and the measurement is stable when at least three successive readings differ by less than a predefined value.

The signal generated by the photodiode is formed from two components:

The term of interest is V_ref|ected while the term Vdiffused might cause a constant offset that limits the interval of useful signal. To limit this negative effect, said offset can be eliminated by means of said optional dedicated circuit 14a by the microprocessor 1 1 in order to report, in the optimum measurement interval, the useful signal produced by the photodiode 12.

According to a further aspect of the invention, it is preferable for the light of the LEDs to be suitably modulated with a frequency around KHz, in order to make the system insensitive to the ambient light. This necessitates the use of a software or hardware demodulator to regain the correct measurement of the reflected light after removing the constant contribution of the ambient light.

Switching-on of the LEDs is controlled by a suitable circuit 17 controlled by the microprocessor 11.

A preferred calibration procedure envisages the following steps:

- starting the calibration procedure, for example by pressing at least one key on keypad 16, or by a code of notches or holes on the strip, so that the microprocessor is readied for calibration,

- next, the microprocessor shows the instructions on the display that the user must carry out and in particular requests sequential insertion of a predefined strip number with colour corresponding to known, stable concentration, for example made with suitable paints or by strip impregnated with coloured liquids that simulate a known concentration,

- then, for each strip inserted, the firmware calculates the coefficients of logarithmic regression in order to correlate the measured reflectance with the bilirubin level.

According to another preferred variant of the reader, shown in Fig. 6, similarly to the preceding version, the reflectometer comprises a housing 30, entirely similar to Fig. 5 and indicated there as 10 and shown in Fig. 4, comprising said diode with blue light 33 and a photodiode 32 inserted near a groove 30'. An LED 38 with green light is optional. Switching-on of the LEDs is controlled by a suitable circuit 37 connected to a USB interface 31 . The following are connected to the same USB interface 31

- an analogue/digital converter 36, able to convert the signals generated by the amplification section 35, 35' and 35" of the signals generated by the LEDs and

- an optional compensating circuit of the offset 35 described above.

Electrical supply of the reader thus described, control of its components as well as filtering and display of the results is performed by a PC computer, not shown, connected to the USB port of said interface 31 and comprising suitable software. Stabilization and conversion of the electrical energy supplying the device is managed by a suitable optional circuit 38.

The present variant is particularly advantageous in countries and locations where a computer is available, and the electric power supply for the latter, while the previous variant is more advantageous in poor countries, since the device comprises per se the necessary components for performing a bilirubin measurement.

Both variants work similarly, employing the same methods of operation, calibration and measurement of bilirubin, and they only differ in that the microprocessor 1 1 , and the man/machine interface, as well as the power is/are performed/supplied by a computer 98.

The system can measure plasma bilirubin with an accuracy of ±5% at bilirubin concentrations in the range 3-30 mg/dL (approx. 50-500 μΜοΙ/L) at haematocrit level up to 60%. Even at high haematocrit levels (up to 75%), 20-25 μΙ of blood is sufficient for measuring plasma bilirubin.

The accuracy of a system as described was determined using both haematocrit (hct 45%, bilirubin 9.2 mg/dL) and plasma (bilirubin 5 mg/dL, 22 mg/dL). The coefficient of variation (CV) calculated on 9 and 2 samples respectively was less than 5% for both. The reproducibility and accuracy of the system was tested on the blood of adult subjects containing high (55-60%) and low haematocrit concentrations (40-45%) and containing bilirubin levels of 25-30 mg/dL and 8-10 mg/dL.

The results produced by the system were compared with two methods approved for measuring total bilirubin using plasma prepared by centrifugation from the same samples (UB Analyzer, Arrows, Jendrassik Graff).

The observed discrepancies were approx. 8% (slightly higher for high bilirubin levels and slightly lower for low bilirubin levels). Thus, these results are comparable to the results obtained in the laboratory.

The reading time can vary from 12 seconds with haematocrit concentration of 25% up to 150 seconds for haematocrit concentrations of 65%, depositing 25 μΙ of blood on a filter of about 50-54 mm² or depositing 20 μΙ of blood on a filter of about 42-46 mm². Using these dimensions, advantageously, the contamination of the blood is limited when the haematocrit exceeds a concentration of 60-62%.

Thus, it is clear that on reducing the dimensions of the filter, the diffusion time is reduced, but there is a risk that the membrane will be contaminated by the corpuscular portion of the blood, for concentrations especially at haematocrit levels above 60%. Therefore the filter dimensions must be greater than 40 mm².

With the aid of Figs. 7 to 8, a method of operation of the reflectance reader is described, independently of the variant implemented.

The method comprises the following steps:

- step 41 checks whether a calibration of the device is stored in memory,

- if Yes (step 41 yes), step 42 checks that the voltage level supplied from the power supply 88, 98 is adequate, if the voltage level is not adequate it stops, step 53, otherwise it goes to the next step - step 43, wait for insertion of a strip in the reader (1 , 1 '),

- step 44, reading by switching-on the blue LED 13, 33, optional loading of any value of offset relative to the blue LED memorized during calibration of the reader and subtraction, at the analogue stage, of said value of offset from the output signal of the photodiode 12, 32,

- step 45, waiting until the reading has stabilized with optional setting of a time-out, for example of 350 s,

- step 46, carrying out a predefined number of readings, preferably 256,

- step 47, switching-off the blue LED and optional reading by switching-on the green LED 13, 38 with optional loading of the value of offset relative to the green LED memorized during calibration of the reader and subtraction, at the analogue stage, of said optional value of offset from the output signal of the photodiode 12, 32,

- step 48 waiting for stabilization of the measuring circuit: this stabilization is necessary to stabilize the electronic circuitry defining the reader, to stabilize the conversions of the analogue/digital signal in relation to optional elimination of the offset and to obtain a stable light from the LEDs, which must reach an optimum operating temperature,

- step 49, performing a predefined number of readings, preferably 256.

- step 50, calculation of the threshold of the green from the measurement of the blue by means of a conversion table that links at least one reflectance value of bilirubin with at least one reflectance value of haemoglobin resulting from lysis of the red blood cells,

- step 51 , verification that the measurement made with the green LED is below a predefined threshold,

- if Yes (step 51 yes) step 52, cancellation of the measurements and sending of a warning message,

- step 53, end,

- if the calibration is not performed (step 41 No),

- step 54, carrying out calibration of the reader, and

- step 42 checking battery levels, etc. - if the measurement carried out with the green LED is above said predefined threshold (step 51 No), step 56, calculation of the bilirubin concentration by means of the coefficients of the calibration curve stored in memory,

- step 57, presentation of the results on display 16'.

It is preferable for subtraction of any value of offset from the reflectance measurement to be effected at the hardware level, i.e. in the analogue domain and not digital because otherwise the amplifier stages might saturate with unforeseeable results. An optimum number of readings is found to be 256 because 256 is equivalent to 2^Λ8, which is the maximum number of values that can be expressed by one byte (8 bits). Using this value, the routine for calculating the mean value becomes very quick and efficient.

The 256 readings are necessary for filling the delay line of the FIR filter (namely 256 taps) and to be able to calculate at least one output. Moreover, filter design is simplified since division by 256 coincides with shift of 8 bits to the right.

According to the present embodiment, in which the variable sum is an integer without sign at 32bit, there would be space for performing 2^Λ20 =1048576 (because 32- 12=20bit) readings added together then carrying out 16 shifts to the right (division by 65536) obtaining a 16-bit result for utilizing the so-called "Process Gain" of interpolation, which represents the basic principle of the sigma-delta converters. Implementation of this method of signal processing makes it possible to obtain higher resolution or use 10-bit or 8-bit A/D converters, reducing the costs of the reader, but maintaining a final accuracy equivalent to 12-bit and a pass band equally limited, for reducing noise.

Fig. 8 shows a flow chart relating to a method of calibration of the reader performed at step 54 of the method of Fig. 7.

This method comprises the following steps:

- step 70 up to step N+70 carrying out a sequence of readings of a coloured membrane by means of a known coloration determining a reflectance value relating to a known bilirubin titre,

- step 72, execution of a logarithmic regression on said sequence of readings and calculation of the coefficients of the logarithmic function applied in the calculation of the bilirubin concentration described at step 56,

- storage of the coefficients in the memory of the microprocessor 1 1 or in the memory of the computer to which the reader is connected via USB port.

Fig. 7, top right, also shows a flow chart of the method of checking that the voltage level supplied by the power supply 88, 98 is adequate.

Rather than logarithmic regression, a different function could also be used, for example contiguous sections of straight lines on suitable intervals.

The present invention can advantageously be implemented by means of a computer program that comprises coding means for performing one or more steps of the method, when this program is carried out on a computer. Therefore it is intended that the scope of protection should extend to said computer program and moreover to computer-readable means that comprise a recorded message, said computer- readable means comprising program coding means for carrying out one or more steps of the method, when said program is carried out on a computer.

The elements and the characteristics illustrated in the various preferred embodiments can be combined with one another, while remaining within the scope of protection of the present application.

Claims

1. Method of measuring total plasma bilirubin of a blood sample of a predefined amount, the method comprising the following steps:

- first irradiation of the plasma with blue light,

2. Method according to claim , further comprising the following steps:

- second irradiation of the plasma with green light,

3. Method according to one of the preceding claims, in which said approximating curve is of the negative logarithmic type or comprises contiguous sections of straight lines on suitable intervals.

4. Method according to one of the preceding claims, comprising the step of modulating said first or said second irradiation at a frequency of the order of kHz.

5. Method according to any one of the preceding claims, further comprising the step of arranging a filter (22, 82) having a surface of 40-55 mm² and of arranging a membrane (23, 83) having a capillarity between 70 and 105 (s/4 cm) and calibre 200 (μΐη) and having a surface of 20-30 mm², and preferably of nitrocellulose, so as to be in contact with the filter, the membrane being capable of absorbing, from the filter, the plasma of said predefined amount of blood when an amount of blood of 20-25 μΙ is deposited on the filter.

6. Method according to claim 5, in which said membrane has an optimum surface of 24 mm² and/or in which said filter has an optimum surface of 48 mm².

7. Method according to either of claims 5 or 6, in which said membrane and said filter partially overlap with an area of overlap between 2 and 6 mm².

8. Method according to any one of claims 5 to 7, further comprising the step of arranging means for constricting and covering (86) of said filter to facilitate the passage of plasma into said membrane (23,83).

9. Strip (2, 80) for separating the plasma from a residual corpuscular portion of a predefined amount of blood, in particular for measuring the total plasma bilirubin, comprising

- a supporting band (21 , 81 );

- a filter (22, 82) able to retain a corpuscular portion of a predefined amount of blood deposited on the filter;

- a membrane (23, 83) able to receive the plasma of said amount of blood from the filter; and

- optionally a containing support (99),

the filter (22, 82) and the membrane (23, 83) being attached to the supporting band (21 , 81) in mutual contact with one another;

the filter (22, 82) having a surface of 40-55 mm² and the membrane (23, 83) having a capillarity between 70 and 105 (s/4 cm) and calibre 200 (μηη) and a surface of 20- 30 mm².

10. Strip according to claim 9, further comprising means for constricting and covering (86) enabling a pressure to be exerted on the filter that facilitates the passage of the plasma of said predefined amount of blood into the membrane.

1 1 . Strip according to claim 9, in which the filter (22, 82) and the membrane (23, 83) are partially mutually superposed, determining an overlap (24, 84) on said supporting band (21 , 81 ).

12. Strip according to claim 1 1 , in which

the supporting band (21 , 81 ) has a length in the range 28-35 mm and a width in the range 3-7 mm and/or,

the width of the membrane and of the filter is 4 mm and said overlap is effected along the edge defining the width of filter and membrane to an extent of 0.5-3 mm.

13. Strip according to claim 12, in which said membrane is of nitrocellulose and/or said filter consists of a matrix of glass fibre and polyester.

14. Reader (1 , 1 ') of strips (2, 80) as defined in claims 9 to 13 for measuring total plasma bilirubin, comprising

- first means of irradiation (13, 33) of said membrane (23, 83) with blue light

- a reflectometer (10, 12, 13, 30, 32, 33) comprising a housing (10', 30') for the insertion of a strip (2, 80) able to perform first measurements of reflectance (12, 32) corresponding to irradiation with said first means of irradiation (13, 33),

- processing means (1 1 , PC) connected to the reflectometer, comprising means for calculating a bilirubin concentration by means of a linearized function approximating a bilirubin absorption curve as a function of its concentration according to said first irradiation.

15. Reader according to claim 4, further comprising

- means for power supply (88, 98) of the reader, processing means (1 1 ) and means for man/machine interface (16, 16') or

- means for interface (31 ) to a communication port of a PC computer supplying the reader (1 '), a PC computer comprising said means for calculating a bilirubin concentration and means for man/machine interface.

16. Reader according to claim 14 or 15, in which said reflectometer comprises a photodiode (12, 32) connected to said processing means (1 1 , PC) by an amplification section (15, 15', 15"', 35, 35', 35"), means for low-pass filtering of the signal generated by said photodiode, external or integrated in said processing means (1 , PC) and in which said first means of irradiation (13, 33) comprise at least one LED diode with blue light.

17. Reader according to claim 16, in which said reflectometer further comprises - second means of irradiation (18, 38) of said membrane (23, 83) with green light, controlled by said switching circuit (17, 37) from said processing means (1 1 , PC) and in which said processing means (1 1 , PC) comprise means for calculating an estimate of the contamination of the plasma by haemoglobin by means of an electrical signal generated by the photodiode (12, 32) when said second means of irradiation are active and/or - a circuit for compensating offset (14, 34), cancelling a portion of signal generated by the photodiode due to capture of diffuse radiation (Vdjff_use), connected to said amplification section (15, 15', 15'", 35, 35', 35") and controlled by said processing means (11 , PC).

18. Reader according to one of claims 14 to 17, further comprising a USB interface (31 ) and a computer (PC) defining said processing means and said means of power supply (98).

19. System of the "point of care" type for measuring total plasma bilirubin comprising

- a strip (2, 80) according to one of claims 9 to 13,

- a reader (1 , V) of strips (2, 80) according to one of claims 14 to 18 operating according to the steps of one of claims 1 to 4.

20. Computer program that comprises program coding means able to perform the steps of claims 1 to 4, when said program is run on a computer.

21. Computer-readable means comprising a recorded program, said computer- readable means comprising program coding means able to perform the steps of claims 1 to 4, when said program is run on a computer.