RU2140083C1 - Method of determination of content of main derivatives of hemoglobin - Google Patents

Abstract

FIELD: medical diagnoses in field of hematology for control of blood gas composition; applicable also in qualitative spectrophotometric analysis of any chemical system with overlapping spectrums of component absorption. SUBSTANCE: content of main derivatives of hemoglobin is determined with use of hemolyzed 1% solutions blood. Measurement of absorption spectrums is carried out by spectrophotometer with six analytic lengths of waves within the band of 450-650 mm. Subsequent calculation of concentrations of oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin and methemoglobin by measured optical density is carried out with the help of program support. Claimed method may be used as basis for development of simple and reliable device. EFFECT: simplified measuring devices and higher accuracy of analysis. 4 cl, 5 dwg

Description

 The invention relates to medical diagnostics in the field of hematology and can be used to control the gas composition of blood in intensive care, toxicology, intensive care, to determine the effect on the gas composition of hemoglobin of environmental factors (environmental conditions, radiation exposure), in space medicine. The method can be the basis for creating a simple and reliable device.

 A known method and device for determining the oxygenation of hemoglobin in optically accessible tissues (US Patent N 5318122, IPC A 61 B 5/00, 6/00).

 For oximetry, a selected tissue region is examined using collimated probe light of at least two specific wavelengths in the hemoglobin absorption band around an isobestic absorption wavelength of about 501 nm. At least one wavelength is close to isobestic and at least one nonisobestic. The reflection of the probe light from the tissue for each wavelength is determined and the signal intensities corresponding to the reflection are provided. In accordance with the intensities of the reflection signals, reflection coefficients adequate for oxygen saturation are determined for isobestic and nonisobestic wavelengths, which represent the oxygenation of hemoglobin.

 The disadvantage of this method is to determine only the degree of oxygenation of hemoglobin, which does not allow for a more complete diagnosis of the patient's condition. In addition, the content of carboxyhemoglobin and methemoglobin is neglected in the calculations, which in some cases significantly reduces the accuracy of oximetry. The method cannot be used in the study of pathological conditions.

 There is also a method of analyzing the gas composition of the blood (US Patent N 5355880, A 61 B 5/00).

The analysis of the gas composition of blood in a living organism is carried out by measuring the intensity of light absorption by the drug under illumination with three different light sources in the range of 500-2500 nm. The magnitude of the light absorption intensity for each of the preparations depends on the pH, the concentration of HCO ₃ ^- , pCO ₂ , pO ₂ and oxygen saturation. After determining at least two values for the pH, HCO ₃ and pO ₂ values, the oxygen content in the analyzed function can be calculated with a high degree of certainty from these absorption intensity values.

 The disadvantage of this method is the determination of only the oxygen content in the analyzed drug, which significantly reduces the information content of this method. The method is not applicable in some pathological conditions.

 A known method and device for direct spectrophotometric measurement in whole unchanged blood (Application PCT (WO) N 94/08237, IPC G 01 N 33/48).

 A method and apparatus for accurate spectrophotometric determination of the concentrations of various types of hemoglobin in whole blood without hemolysis or dilution is proposed.

 To eliminate the complex optical properties of whole blood, the method provides for the use of an optical device 10, 11, 12, 13 and 14, designed to optimize measurements and to determine the true optical density of whole blood and reduce the effect of light scattering on spectrophotometric measurement of the concentration of various constituent components, and correction measurements of hemoglobin concentration taking into account light scattering and the influence of the final bandwidth of predominantly monochromatic light. In the optical device 10-14 (including the optical cuvette 11), all optical parameters, such as the thickness of the sample, the shape and size of the detector, the distance between the sample and the detector, wavelength, monochromaticity, the maximum angle of light capture by the detector, are optimal to reduce the fraction of light scattering in the total optical attenuation of whole unchanged blood and an increase in the fraction of true optical density.

 The disadvantage of this method is the complexity of the measuring equipment, which makes it difficult to use in wide clinical practice.

где

оптические плотности слоя крови;
n = 1,2,3 - индексы, относящиеся к длинам волн λ₁ = 660 нм, λ₂ = 700 нм, λ₃ = 805 нм;

показатели поглощения слоя крови, определяемые из выражения

где

параметры, определяемые по величине коэффициентов диффузионного отражения

одного из слоев крови из соотношения

коэффициент диффузного пропускания слоя крови с толщиной l' = 0.1 см;

коэффициент диффузного пропускания слоя крови с толщиной l''=0.14 см;
l' - толщина слоя крови = 0.1 см;
l'' - толщина слоя крови = 0.14 см;

миллимолярные показатели поглощения метгемоглобина;
a - относительная концентрация метгемоглобина;

миллимолярные показатели поглощения дезоксигемоглобина;
b - относительная концентрация дезоксигемоглобина;

миллимолярные показатели поглощения оксигемоглобина.Closest to the claimed method is a method for determining the relative concentrations of hemoglobin derivatives (copyright certificate 1613955, G 01 N 33/49), including the placement of blood samples in two cuvettes with different optical paths, measuring the diffusion reflection coefficients and the relative transmittance of the two layers, characterized in that, in order to simplify the method, photometry is performed at wavelengths of 660, 700, 805 nm, followed by the calculation of the relative concentrations of meth-, oxy-, and deoxyhemoglobin according to the formula

Where

optical density of the blood layer;
n = 1,2,3 - indices related to wavelengths λ ₁ = 660 nm, λ ₂ = 700 nm, λ ₃ = 805 nm;

blood absorption indicators determined from the expression

Where

parameters determined by the magnitude of the diffusion reflection coefficients

one of the blood layers from the ratio

diffuse transmittance of a blood layer with a thickness l '= 0.1 cm;

diffuse transmittance of a blood layer with a thickness l '' = 0.14 cm;
l '- blood layer thickness = 0.1 cm;
l '' - blood layer thickness = 0.14 cm;

millimolar methemoglobin absorption indicators;
a is the relative concentration of methemoglobin;

millimolar absorption of deoxyhemoglobin;
b is the relative concentration of deoxyhemoglobin;

millimolar absorption of oxyhemoglobin.

c is the relative concentration of oxyhemoglobin;
The advantage of this method is the consideration of diffusion scattering in the calculations, which increases the accuracy of the results; as well as the determination of three hemoglobin derivatives, including methemoglobin, which allows the use of this method for a number of acute poisonings.

 The known method does not provide for determining the content of carboxyhemoglobin, as well as the absolute concentrations of the studied hemoglobin derivatives, which significantly reduces in most cases the information content of the method; in addition, the use of whole blood raises the requirements for measuring equipment.

 The objective of the present invention is to provide a method for determining the content of the main derivatives of hemoglobin, which provides the measurement of the four components of hemoglobin, including carboxyhemoglobin, simplification of measuring equipment and increase the accuracy of analysis.

(1)
где

- оптическая плотность раствора крови на i-й длине волны;
ε $\genfrac{}{}{0ex}{}{\text{λ}}{\text{j}}$ - миллимолярные показатели поглощения j-й производной гемоглобина на i-й длине волны;
c_j - концентрация j-ой производной;
n - число анализируемых производных гемоглобина;
m - число аналитических длин волн;
i = 1, 2, ..., m.The specified technical result is achieved in that in the method for determining the content of the main hemoglobin derivatives based on the photometry of blood samples at several wavelengths of the visible range, characterized in that the determination of the optical density of hemolized one percent blood solutions is carried out at m ≥ n wavelengths selected by linear programming methods and information coefficients, in the range of 450 - 650 nm, the calculation of the concentrations of hemoglobin derivatives, in particular oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, methemoglobin is carried out according to known molar absorption coefficients from a system of equations of the form:

(1)
Where

- the optical density of the blood solution at the i-th wavelength;
ε $\genfrac{}{}{0ex}{}{\text{λ}}{\text{j}}$ - millimolar absorption indices of the j-th hemoglobin derivative at the i-th wavelength;
c _j is the concentration of the jth derivative;
n is the number of analyzed hemoglobin derivatives;
m is the number of analytical wavelengths;
i = 1, 2, ..., m.

 The overdetermined system (1) is solved using specially created software, including least squares (LSM) methods, linear programming (MLP), algebraic background correction (ACF), and combined methods including MLP and ACF.

 The method is illustrated by graphs and tables, in which Fig. 1 shows the spectral dependence of the millimolar absorption coefficients of the main hemoglobin derivatives (1-hydroxyhemoglobin, 2-deoxyhemoglobin, 3-carboxyhemoglobin, 4-methemoglobin); Fig. 2 shows the degree of the content of the main hemoglobin derivatives nicotine intoxication for men (1 - non-smoking patient, 2 - smoking patient); figure 3 shows the dependence of the sum of the calculated concentrations of hemoglobin derivatives on the value of the total hemoglobin concentration determined by the standard cyanmethemoglobin method.

 The method for determining the content of the main derivatives of hemoglobin is as follows.

The study used heparinized blood samples taken from the ulnar vein. In order to avoid loss of red blood cells, centrifugation was not performed. A 1% blood solution was prepared as follows: 0.06 ml of a 0.04% ammonia solution (to clarify the solution) and 0.5 ml of blood were added to 20 ml of distilled water, after 1-2 minutes (after hemolysis) 25 ml of 0.0667 M KH ₂ PO ₄ /0.0667 were added. M Na ₂ HPO ₄ • 2H ₂ O, pH 7.2. The total volume of the solution was adjusted with distilled water to 50 ml. The absorption spectra were recorded at six wavelengths in the range 450–650 nm in a quartz cuvette with an optical path length of 1.001 cm on an SF-20M double-beam spectrophotometer with automatic adjustment of the slit width. As a comparison solution, the above buffer solution was used. Measurements were taken within 1 hour after blood sampling.

где i = 1, 2, ..., m;
m - число аналитических длин волн;
n - число определяемых компонентов;

молярные коэффициенты поглощения (м.к.п.) для j-го компонента на длине волны λ_i;
c_j - определяемая концентрация j-го компонента;

оптическая плотность на длине волны

Из-за сложности объекта исследования (перекрывание спектров анализируемых производных, практически полное подобие спектров оксигемоглобина и карбоксигемоглобина, число компонент равное четырем) применение простых спектрофотометрических методов анализа не приводит к необходимой точности.The calculation of the content of hemoglobin derivatives is reduced to solving the system of Fierordt's equations:

where i = 1, 2, ..., m;
m is the number of analytical wavelengths;
n is the number of determined components;

molar absorption coefficients (lmc) for the j-th component at a wavelength of λ _i ;
c _j is the determined concentration of the jth component;

optical density at a wavelength

Due to the complexity of the object of study (overlapping spectra of the analyzed derivatives, almost complete similarity of the spectra of oxyhemoglobin and carboxyhemoglobin, the number of components is four), the use of simple spectrophotometric methods of analysis does not lead to the necessary accuracy.

 The problem can be solved by going over to overdetermined systems of linear algebraic equations (i.e., SLAE with m> n).

 There are various methods for resolving overridden SLAEs. These methods are distinguished by the criterion to which the resulting solution should best fit. Different solutions obtained using different criteria can fundamentally differ from each other. Calculations by these methods are very laborious and time consuming. In order to overcome these difficulties and improve the method, special software has been created, including least squares (LSM), linear programming (MLP), algebraic background correction (ACF) methods, and combined methods including MLP and ACF.

Элемент R_ij показывает, какую долю информации несет i-я линия при определении j-й компоненты смеси. Чем выше информационные коэффициенты, тем меньше погрешности найденного решения. Также при определении аналитических длин волн использовались разности вида

и отношения вида

с целью выбора точек максимального различия в спектрах оксигемоглобина и карбоксигемоглобина (из-за причин, указанных выше). Затем полученный набор длин волн исследовался с помощью МЛП. Состав набора варьировался в процессе анализа. Длины волн, не оказывавшие влияния на результат, удалялись из набора. Еще одним критерием выбора являлся минимум разности между экспериментальным и редуцированным спектром на определенной длине волны.The analysis used millimolar absorption indices (the work of Sigaard-Andersen J., Norgaard-Pedersen O.V., Rem 1. (1972) Hemoglobin pigments II: Photometer for oxygen saturation, carboxyhemoglobin, and methemoglobin in cappilary blood, in Clinica; (Chimica Acta), 42, pp. 101-108) shown in FIG. 1. The choice of analytical wavelengths was carried out using the method of information coefficients. To assess the optimality of the set of analytical wavelengths and the magnitude of the analysis error, a matrix of information coefficients is constructed, consisting of elements of the form:

The element R _ij shows how much information the i-th line carries in determining the j-th component of the mixture. The higher the information coefficients, the less the error of the solution found. In determining the analytical wavelengths, differences of the form

and relationships of the kind

in order to select points of maximum difference in the spectra of oxyhemoglobin and carboxyhemoglobin (due to the reasons mentioned above). Then the obtained set of wavelengths was investigated using MLP. The composition of the kit varied during the analysis. Wavelengths that did not affect the result were removed from the set. Another selection criterion was the minimum difference between the experimental and reduced spectra at a certain wavelength.

 Testing of the method was carried out on solutions with a known content of components. Three-component mixtures containing cobalt, chromium and nickel chlorides, which have overlapping absorption spectra, were used as reference solutions. Comparative results are given in table 1.

A good agreement between the results of the analysis and the reference values indicates the effectiveness of the method and indicates the possibility of studying complex solutions with overlapping absorption spectra of the components. The gas composition of whole blood solutions was studied using software on the absorption spectra of multicomponent mixtures of hemoglobin derivatives containing HbO ₈ , Hb, Hb (CO) ₄ , MetHb. The results are presented in table.2.

 To illustrate the developed technique, the dependence of the concentrations of hemoglobin derivatives on the degree of nicotine intoxication for men was obtained. The results are shown in figure 2, which clearly shows the negative changes in the human body (a decrease in the content of oxyhemoglobin due to an increase in the concentration of carboxyhemoglobin, and as a result of this - a decrease in the degree of oxygenation of hemoglobin).

 An experimental assessment of the accuracy of the developed method for determining the content of the main hemoglobin derivatives was carried out. The criterion for the accuracy of calculating the concentrations of hemoglobin derivatives was the coincidence of the sum of the concentrations of hemoglobin derivatives calculated by this method with the value of the total hemoglobin concentration in the same blood sample as determined by the standard cyanmethemoglobin method. Figure 3 shows the dependence of the calculated sum of the concentrations of hemoglobin derivatives on the value of the total hemoglobin concentration. The slope coefficient of the approximating straight line is 0.997, the accuracy of the approximation is 0.987, which confirms the good agreement of the results.

 The developed method is applicable to the quantitative spectrophotometric analysis of any chemical systems with overlapping absorption spectra of the components. Based on the proposed method, a specialized multi-wavelength module with a fixed set of analytical wavelengths for use in clinical practice can be created.

Example. A blood solution prepared by the above method was placed in a cuvette. Measurements at analytical wavelengths showed the following results for optical density: D ¹ = 1.308; D ² = 0.4696; D ³ = 1.0234; D ⁴ = 0.9128; D ⁵ = 0.7853; D ⁶ = 1.075.

С помощью программного обеспечения произведен расчет содержания окси-, дезокси-, карбокси- и метгемоглобина и получены следующие результаты в единицах г/100 мл (в %):

10.9319 (79.50); C_Hb = 2.3828 (17.38);

= 0.2576 (1.93); C_MetHb = 0.1610 (1.19).Millimolar absorption coefficients used for each length in l / (mmol • cm):

Using software, the oxy-, deoxy-, carboxy- and methemoglobin content was calculated and the following results were obtained in units of g / 100 ml (in%):

10.9319 (79.50); C _Hb = 2.3828 (17.38);

= 0.2576 (1.93); C _MetHb = 0.1610 (1.19).

Claims (4)

1. A method for determining the content of the main hemoglobin derivatives, based on photometry of blood samples at several wavelengths of the visible range, characterized in that photometry is carried out at m≥n wavelengths in the range of 450 - 650 nm, the optical density of the blood solution is measured at selected wavelengths and determine the concentration of derivatives according to known molar absorption coefficients from a system of equations of the form:

Where

optical density of a blood solution at the i-th wavelength;

millimolar absorption indices of the j-th hemoglobin derivative at the i-th wavelength;
with _j is the concentration of the jth derivative;
n is the number of analyzed hemoglobin derivatives;
m is the number of analytical wavelengths;
i - 1, 2 ..., m.  2. The method according to claim 1, characterized in that four hemoglobin derivatives are determined: oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, methemoglobin.  3. The method according to claim 1, characterized in that they use a 1% solution of blood, including distilled water, 0.04% ammonia and a buffer solution.  4. The method according to p. 1, characterized in that the choice of wavelengths is carried out by linear programming methods and information coefficients.

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