WO2016071887A1

WO2016071887A1 - Method to measure glycated hemoglobin

Info

Publication number: WO2016071887A1
Application number: PCT/IB2015/058636
Authority: WO
Inventors: Paolo Galiano
Original assignee: Alifax S.R.L.
Priority date: 2014-11-07
Filing date: 2015-11-09
Publication date: 2016-05-12

Abstract

Method to measure glycated hemoglobin by means of fluorometry carried out on a blood sample, which provides the following steps: - preparing a fluorescent marker, exciting it by emitting radiation to emit fluorescence, and recording the fluorescence only of the fluorescent marker (Fblank); - associating the blood sample to the fluorescent marker to obtain a solution, in which the fluorescent marker and the blood sample are combined at a zero time; - exciting the solution with the radiation and detecting the resulting fluorescence as the reaction between glycated hemoglobin and fluorescent marker progresses; - calculating, from the values of fluorescence detected, the value F0, which represents the fluorescence of the solution at zero time when the blood sample is introduced and the glycated hemoglobin begins to bind with the fluorescent marker, and the value Finf, which represents the fluorescence at infinite time, when all the glycated hemoglobin has reacted with the fluorescent marker; - calculating, from the values of F0 and Finf, the variation in fluorescence during the reaction occurring in the first step; and - determining, from the values calculated, directly, that is to say, without calculating the value of total hemoglobin, the percentage of glycated hemoglobin with respect to the total hemoglobin present in the blood sample at zero time before the reaction with the fluorescent marker.

Description

"METHOD TO MEASURE GLYCATED HEMOGLOBIN"

FIELD OF THE INVENTION

The present invention concerns a method to measure glycated hemoglobin in the blood. In particular the present invention concerns a method to measure glycated hemoglobin by mixing it with a fluorescent marker, and subsequently measuring the fluorescence with a fluorometer.

BACKGROUND OF THE INVENTION

It is known that hemoglobin is the main constituent of red corpuscles, and is used to transport oxygen in the blood to all the tissues of the organism. Hemoglobin is present in different variants, 90% of which are represented by so- called "hemoglobin A". Hemoglobin A is formed by two pairs of chains of amino acids, called "alpha chain" and "beta chain".

The glucose present in the blood can bind covalently with a terminal of the beta chain in a non-enzymatic process, forming the so-called glycated hemoglobin or HbAlc. This reaction, called "glycation", gives rise to a bulkier protein than hemoglobin A, and with reduced capacity of transporting oxygen, with consequent reduced oxygenation of organs and tissues in the human body. The percentage of glycated hemoglobin is directly proportional to the quantity of glucose in the blood and, considering the irreversibility of the glycation reaction, the HbAlc contained in the red corpuscles circulates in the blood for the entire duration of their life. Since the average life of red corpuscles is comprised between 90 and 120 days, measuring the ratio between the quantity of glycated hemoglobin and total hemoglobin allows to evaluate the average quantity of glucose present in the blood in the 2-3 months prior to measuring.

Within certain limits of the glycated/total hemoglobin ratio, for example when it has values comprised between 3% and 6.5%, corresponding to about 20-48 mmol/mol, the glycosylation process of hemoglobin is considered a normal biological function and does not entail any danger for an individual.

To evaluate the correct glucose contribution in the diet, especially for diabetic patients (both type 1 and type 2), dosage of HblAc is especially useful because it allows to monitor a correct diet in the 3 months before the HblAc test is done. This monitoring can therefore be associated with a self-check by the individual, but can also be used to assess the risk of developing the complications of diabetes.

It is also known that, at present, and as recommended by the American Diabetes Association (ADA), the glycated hemoglobin test has become part of the diagnostic criteria for diabetes mellitus, alongside the traditional criteria.

There are recommendations to carry out the glycated hemoglobin test frequently and regularly, so as to keep said value under control, both for patients who are already diabetic, and for those at risk of diabetes, and also for prevention.

The glycated hemoglobin test can be carried out in analysis laboratories, for example at hospitals, using an HPLC or immunological technique.

However, given the need to increase the frequency of the measurements and to put patients at their ease during the test, there is an increasing requirement to use equipment suitable for measuring glycated hemoglobin that is distributed throughout the national territory, inside points of care for example, such as doctor's surgeries and pharmacies, easier to reach for the average patient.

On this point it is known that a suitable method for measuring glycated hemoglobin is the immunological method, which is based on the antigen reaction (glycated hemoglobin) and an antibody bonded with particles of latex. The known reaction of immunological agglutination is used, which can be read with nephelometric methods, light scattering or a turbidimetric method that uses a photometer to detect the absorbance of the aggregate or agglutinate.

Another method used in the state of the art to detect glycated hemoglobin is the enzymatic method, which uses a lysis swab and digestions with proteases; this process releases amino acids, including glycated- valine compounds, from the beta chains of hemoglobin.

The glycated-valine compounds serve as a substrate for the fructosyl valine oxidase (FVO) enzyme.

The FVO enzyme breaks the N-terminal groups of the valine and produces H₂O₂ which in the presence of peroxidases (POD) and a chromogen gives a coloration proportional to %HbAlc. Both methods require the use of a calibration curve at different concentrations of glycated hemoglobin on which to extrapolate the value of glycated hemoglobin of the blood sample examined.

Recently, as shown for example in US-B-7,998,742, a fluorometric method has been proposed, which exploits the great affinity of boronate marked with eosin, as a basic fluorescent substance, with the HbAlc present in a blood sample to be analyzed. The method exploits the peculiarities that boronate has in binding through affinity by means of its oxydryl groups with the oxydryl groups also present on the glycated hemoglobin, liberating H₂O.

This characteristic allows to measure the absolute concentration of HbAlc with a direct reaction and a subsequent calculation of the percentage, after having measured the value of total hemoglobin.

The principle on which this method is based is that the reduction in the fluorescence of the boronate is proportional to the quantity of glycated hemoglobin present in the blood sample, from which it is possible to measure the quantity of glycated hemoglobin in the sample. The reduction in fluorescence is due to the fact that, when the hemolysis of the red corpuscles starts inside the marker/sample mixture, the glycated hemoglobin which reacts with the oxydryl groups of the eosin boronate is released.

The method described in US-B-7,998,742 provides to initially record, for example for 2-3 seconds, the basic fluorescence associated only with the fluorescent marker; subsequently, at zero time (F0), the blood sample is joined with the marker and then the fluorescence of the corresponding substance formed (F0 - Finf) is detected.

Afterward, at regular intervals of time, the fluorescence of the marker/sample mixture is measured until it is detected that it has reached an asymptotic value measured at an infinite time.

The values Fb, F0 and Finf, measured during the reaction between the boronate reagent and blood sample, are necessary to calculate FOD and SQ.

The first (FOD = Fluorescence Optical Density) corresponds to the decimal logarithm of the fluorescence of the marker correlated to that of the substance at zero time. The second (SQ = Specific Quenching) corresponds to the difference between the fluorescence at zero time of the substance and fluorescence at infinite time correlated to fluorescence at zero time of the substance.

Each of the two parameters indicated above is proportional, respectively, to the quantity of total hemoglobin and glycated hemoglobin according to a linear development described by corresponding calibration lines and equations of the lines.

Subsequently, the percentage of glycated hemoglobin is identified in terms of ratio between the quantity of glycated hemoglobin with respect to the quantity of total hemoglobin.

One disadvantage of this method is that the percentage of resulting glycated hemoglobin is subject to errors associated both with the construction of the calibration line for glycated hemoglobin, and also with that for the total hemoglobin, since they are calculated separately and therefore the respective errors are added to each other. Furthermore, other little errors could be caused by using lyophile calibrators while the measuring method is on whole blood.

In particular, US '742 provides that the calibration curves are constructed using samples with glycated hemoglobin and total hemoglobin at a known concentration, putting the value of the intercept at zero. This calibration method can lead to imprecisions due to differences between the various batches of raw material, for example eosin boronate, which are used to produce the reagent. Furthermore, the intercepts of the batches produced can be different from each other.

The differences in concentrations and/or possible contaminations of other substances among the different batches of reagent normally come inside nominal intervals supplied by the production Company.

These differences determine a constant variation, that is, an offset, in the measurement of the fluorescent marker, which can cause a change in the parameters of the calibration curve and which therefore also affects the measurement of the percentage of glycated hemoglobin.

These differences, which do not normally represent an obvious problem, in this specific case can be very important for a precise and correct measurement of the percentage of glycated hemoglobin. Indeed, it has been confirmed that the contribution given by a batch of reagent reaches values of 2% more or less with respect to the value of glycated hemoglobin on total hemoglobin.

Although this value is negligible in fluorescence measurements in general, it is not so in this specific case, where the glycated hemoglobin measured generally assumes values comprised between 3% and 6.5%.

In this way, the percentage of glycated hemoglobin resulting from the calculation according to this method can diverge even by 20-25% with respect to its real value measured using the laboratory methods described above and in this context used as reference.

One purpose of the present invention is therefore to perfect a method to measure glycated hemoglobin that allows to identify reliable values and reduces incidence deriving from measuring and calculation errors.

Another purpose of the present invention is to obtain a method to measure glycated hemoglobin that is easy to implement and allows quick analysis and results.

The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.

SUMMARY OF THE INVENTION

The present invention is set forth and characterized in the independent claim, while the dependent claims describe other characteristics of the invention or variants to the main inventive idea.

The present invention concerns a method to measure glycated hemoglobin by means of fluorometry carried out on a blood sample which can provide the following steps:

- conducting a reaction in solution between a blood sample, of which the glycated hemoglobin present therein is to be measured, and a fluorescent marker, in which the fluorescent marker and the blood sample are combined at a zero time;

- exciting the fluorescent marker to emit fluorescence, in which the nature of the fluorescent marker and the nature of the emission are such that the fluorescence occurs at a wave length at which the fluorescence can be altered by the reaction of the fluorescent marker with the glycated hemoglobin; - detecting the resultant fluorescence as the reaction progresses;

- calculating, from the values of fluorescence detected, the value F0, which represents the fluorescence at zero time, and the value Finf, which represents the fluorescence at infinite time, when all the glycated hemoglobin has reacted with the fluorescent marker;

- calculating, from the values of F0 and Finf, the variation in fluorescence during the reaction occurring in the first step; and

- directly determining, from the values calculated and using suitable calculation algorithms, the percentage of glycated hemoglobin with respect to the total hemoglobin present in the blood sample at zero time before the reaction with the fluorescent marker.

This procedure therefore allows to obtain the value of the glycated hemoglobin in percentage terms, without needing to calculate the value of the total hemoglobin, thus reducing possible errors that occur in known procedures.

In one form of embodiment, the algorithm is in the form y = mx + q, where m is a constant value that represents the slope of the straight line and x is defined by [(F0 - Finf)/F0] / [Log (Fblank/FO)], and q represents the contribution of the fluorescent marker that varies according to the batch of reagent.

Advantageously, the method according to the present invention provides to determine the percentage of glycated hemoglobin present at zero time in the blood sample by means of a single calibration line, which is generated on each occasion for each batch of reagent, in this way reducing the calculation errors and approximations associated with using multiple algorithms pertaining to the measurement of the total Hb.

According to the invention, the method allows to eliminate the contribution of differences between the batches of reagent, increasing by even up to 25% the precision of the measurement compared with known systems, including the one in US '742. The fluorescent marker can advantageously consist of eosin boronate, which has great chemical affinity with glycated hemoglobin.

The link between glycated hemoglobin and eosin boronate can be based on their affinity by means of reaction of the glycated hemoglobin with the oxydryl groups of the eosin boronate. The fluorometric method according to the present invention, in substantially the same way as in the state of the art described, for example in US-B-7,998,742, can provide to introduce inside a container a determinate quantity of fluorescent marker, for example eosin boronate, containing a lysant, defining the substance that functions as a reagent blank of the analysis.

Subsequently, it provides to excite the reagent blank with an exciting radiation having a wavelength mating with the type of fluorescent marker used, for example in this specific case of eosin boronate, 520 nm.

The radiation that passes through the reagent blank excites the eosin boronate with energy fall and consequent re-introduction at a different wavelength.

In particular, in one form of embodiment, the exciting radiation can be initially generated by a led that can be directed against a narrow bandpass filter in order to let only the wavelength associated with 520 nm pass.

In one form of embodiment the exposure time of the reagent blank to the exciting radiation can be about 2-3 seconds.

The corresponding emission of fluorescence is subsequently recorded after passage on a second narrow bandpass filter at 544 nm.

The values recorded are converted into an electric quantity, for example nanoampere (nA).

Subsequently, the blood sample to be analyzed can be added to the reagent blank at a zero time. The red corpuscles, reacting with the lysant, can cause the release of the glycated hemoglobin.

The glycated hemoglobin, with affinity to the fluorescent marker as described above, bonds due to said affinity with the reagent eosin boronate, causing a reduction in fluorescence.

The reduction in fluorescence is proportional to the quantity of glycated hemoglobin present in the blood sample analyzed.

The reduction in fluorescence can follow a hyperbolic trend until it reaches an asymptotic value of fluorescence, defined in an infinite moment of time, in which all the glycated hemoglobin is bonded with the fluorescent marker and therefore the reduction in fluorescence terminates.

The fluorometric method according to the present invention provides to record the fluorescence values for each blood sample, in particular those associated with the reagent blank (Fblank), the mixture at zero time (FO), i.e. at the moment the blood sample is introduced, and the mixture at infinite time (Finf) as described above.

Afterward, the values can be processed by calculating known fluorescence parameters, such as FOD (Fluorescence Optical Density) and SQ (Specific Quenching).

These values are respectively identified by the following formulas:

FOD = Log (Fblank/FO) Equation 1

SQ = (FO - Finf)/F0 Equation 2;

In particular, FOD represents the alteration in initial fluorescence due to the union of the blood sample and the fluorescent marker.

SQ represents the alteration in fluorescence of the substance with respect to zero time.

Furthermore, the glycated hemoglobin and the total hemoglobin are proportional respectively to the parameters SQ and FOD cited above.

Consequently, it is possible to express the quantity of glycated hemoglobin and total hemoglobin using equations 3 and 4 as follows:

HbAlc = kl * SQ Equation 3

HbTot = k2 * FOD Equation 4

Therefore the percentage of glycated hemoglobin can be calculated with respect to total hemoglobin by comparing the two quantities using the following equation:

%HbAlc = (Hb A 1 c/HbTot) * 100 Equation 5

where %HbAlc, HbAlc and HbTot indicate respectively the percentage of glycated hemoglobin, the quantity of glycated hemoglobin and the quantity of total hemoglobin.

By substituting the content of equations 3 and 4 in equation 5, it is possible to obtain a different formulation of percentage of glycated hemoglobin represented in the following equation expressed only as a function of SQ and FOD:

%HbAlc = (kl * SQ / k2 * FOD) * 100 =

= (kl / k2) * (SQ / FOD) * 100 Equation 6 In particular, equation 6 is obtained by ordering the factors in its first formulation separating the constant factors, kl and k2, from the factors dependent on the fluorescence, i.e. SQ and FOD.

Therefore, referring to the traditional equation of a line expressed as y = mx + q, it is possible to connect equation 6 to the equation of a straight line.

In particular it is possible to consider that kl/k2 define the angular coefficient of the line (m), SQ/FOD define a sole unknown value x equal to [(F0 - Finf)/F0]/ [Log (Fblank/FO)], and y is the percentage of glycated hemoglobin.

The intercept q can be calculated afterward graphically.

The intercept q mainly represents the constant contribution, or offset, obtained in the different batches of reagent.

For the construction of the calibration curve, the method according to the invention provides to use two calibrators with known glycated hemoglobin values, wherein for each calibrator the SQ/FOD value is automatically measured by the instrument. More exactly, for each calibrator, the instrument measures the SQ/FOD value and traces the straight calibration line with the two values of glycated hemoglobin (HbAlc%) as a function of the respective SQ/FOD values.

According to the invention, the oscillation of the experimental data measured of the values of glycated hemoglobin (HbAlc%) as a function of the respective SQ/FOD values does not significantly influence the linear development that binds these quantities.

Therefore, in order to use the minimum number necessary of calibrators to trace the calibration line, two calibrators are used with known values of glycated hemoglobin.

Returning onto the instrument the value of glycated hemoglobin as a function of the SQ/FOD ratio determined, a calibration curve is automatically obtained on which to determine the values of the samples examined. In particular, the percentage values (%) of glycated hemoglobin of the samples examined are calculated on said calibration curve as a function of the specific SQ/FOD value, thus reducing to a minimum any possible errors, however minimal, in the dispensation of the sample.

The precise measurement for each individual batch of reagent allows to eliminate from the measurements systematic errors not due to the specific quantity of glycated hemoglobin (HbAlc%), but only due to the differences between the batches of reagent.

Therefore, for each new batch of reagent, the invention provides to recalibrate the measuring instrument. In this way, it is not necessary to know the value of total hemoglobin, and also the value of the intercept q is the one found, and depends on the system used and the characteristics of the specific reagent.

Determining the intercept q for every new batch of reagent allows to eliminate from the measurements those imprecisions due to the differences between the individual batches of reagent, which have a variation even up to 25% of the HbA 1 c% measurement.

Advantageously, as we said, the possibility of using a single line to obtain the percentage of glycated hemoglobin directly, i.e. without needing to calculate the value of total hemoglobin in advance, allows to reduce errors associated with using a plurality of calibration algorithms.

It is clear that modifications and/or additions of parts may be made to the method to measure glycated hemoglobin as described heretofore, without departing from the field and scope of the present invention.

Claims

1. Method to measure glycated hemoglobin by means of fluorometry carried out on a blood sample, said method providing the following steps:

- preparing a fluorescent marker, exciting it by emitting radiation to emit fluorescence, and recording the fluorescence only of the fluorescent marker

(Fblank), in which the nature of the fluorescent marker and the nature of the radiation are such that the fluorescence occurs at a wave length at which the fluorescence can be altered by the reaction of the fluorescent marker with the glycated hemoglobin;

- associating the blood sample to the fluorescent marker to obtain a solution, in which the fluorescent marker and the blood sample are combined at a zero time;

- exciting the solution with the radiation and detecting the resulting fluorescence as the reaction between glycated hemoglobin and fluorescent marker progresses;

- calculating, from the values of fluorescence detected, the value F0, which represents the fluorescence of the solution at zero time when the blood sample is introduced and the glycated hemoglobin begins to bind with the fluorescent marker, and the value Finf, that represents the fluorescence at infinite time, when all the glycated hemoglobin has reacted with the fluorescent marker;

- determining, from the values calculated, directly, that is to say, without calculating the value of total hemoglobin, the percentage of glycated hemoglobin with respect to the total hemoglobin present in the blood sample at zero time before the reaction with the fluorescent marker,

characterized in that it also provides to:

- calculate the FOD (Fluorescence Optical Density) and SQ (Specific Quenching) parameters, using the following formulas:

FOD = Log (Fblank/FO) Equation 1

SQ = (F0 - Finf)/F0 Equation 2;

HbAlc = kl * SQ Equation 3

HbTot = k2 * FOD Equation 4 - calculate the percentage of glycated hemoglobin that can be calculated with respect to the total one using a ratio between the two quantities using the following equation:

%HbA 1 c = (HbA 1 c/HbTot)* 100 Equation 5

- substitute the content of equations 3 and 4 in equation 5, to directly obtain the percentage of glycated hemoglobin represented in the following equation expressed only as a function of SQ and FOD:

%HbAlc = (kl * SQ / k2 * FOD) * 100 =

= (kl / k2) * (SQ / FOD) * 100 Equation 6,

wherein the construction of a calibration curve is provided using two calibrators with known glycated hemoglobin values and a straight calibration line is traced with the two values of glycated hemoglobin (HbAlc%) as a function of the respective SQ/FOD values.

2. Method as in claim 1, characterized in that the construction of a calibration curve is carried out for every batch of reagent.

3. Method as in claim 1, characterized in that eosin boronate is used as fluorescent marker.

4. Method as in claim 1, characterized in that a lysant is also introduced into the solution.

5. Method as in claim 3, characterized in that the radiation emitted toward the eosin boronate is generated by a led directed against a filter with a pass band at 520 nm.

6. Method as in claim 5, characterized in that the exposure time to the radiation of the fluorescent marker alone is 2-3 seconds to detect the Fb value.

7. Method as in claim 6, characterized in that the relative emission of fluorescence is recorded after the passage on a second filter with a pass band at 544 nm.