RU2300771C2 - Method for determination of hemoglobin in biological fluids - Google Patents

Abstract

FIELD: medicine, analytical biochemistry.

SUBSTANCE: invention relates to laboratory methods for determination of hemoglobin. Method is carried out by addition of a mixture of concentrated acetic and nitric acid in the ratio 1:1 by volume to control and experimental cuvette followed by addition of a biological fluid sample to be analyzed, for example, whole blood or its dried sample, urine or saliva and the following addition of egg albumin solution and hydrogen peroxide in experimental cuvette and addition of chlorpromazine in the concentration 2.5 mg/100 ml of solution in both cuvettes. Then increase of optical density value of solution is recorded in experimental cuvette against optical density value in control cuvette at wavelength in the range 530-550 nm. Using the proposed method provides enhancing sensitivity and precision of hemoglobin assay in biological fluids, decreasing consumption of used reagents and toxicity degree based on using microamounts of chemically aggressive reagents.

EFFECT: improved method for determination.

5 cl, 2 tbl, 2 dwg, 4 ex

Description

The invention relates to clinical medicine, disaster medicine, obstetrics and gynecology, prenatal medicine, normal and pathophysiology, veterinary medicine.

A known method for determining the concentration of hemoglobin in blood samples by the hemichromic method [patent RU No. 02159442, G01N 33/72, 33/52, publ. 11/20/2000. Bull. No. 32; RF patent No. 1386901, G01N 33/48, publ. 01/15/90. Bull. No. 2; RF patent No. 2054173, CL G01N 33/48, publ. 02/10/96. Bull. No. 4; US 95 524128. 08/31/95, G01N 33/52; US 94275466. 07/14/94, G01N 33/72] by adding 5 ml of a transforming solution to 20 μl of blood and converting normal hemoglobin to the hemichromic form. The optical density of the solution is measured at λ = 540 nm on a calibrated, calibrated instrument (photoelectrocolorimeter) in a 10 mm thick cuvette having zero absorption of distilled water relative to the same control.

The disadvantages of this method include the low sensitivity and, as a consequence of the foregoing, the inability to perform analysis in blood samples of insignificant volume, as well as in samples of dry blood, which, as experience has shown, is required for the prompt diagnosis in a time-deficient environment typical of medicine and emergency veterinary medicine, sports physiology and blood stain identification during forensic examinations. In addition, the determination of hemoglobin by the hemichromic method due to its low sensitivity is limited in blood tests in other biological fluids (urine, saliva), which also limits the use of this method in emergency medicine.

Also known is a method for determining the concentration of hemoglobin in blood samples [patent RU No. 2145715, G01N 33/72, G01N 33/49] by measuring the absorption spectra of blood solutions. From a patient's blood sample, a suspension of red blood cells is obtained, which is placed in a cuvette of a spectrophotometer, and the optical density of a suspension of red blood cells (D) is determined at three wavelengths: 635 nm; 805 nm; 1025 nm, for the method [patent RU No. 2140083, G01N 033/52 G01N 033/72] the procedure is carried out on a spectrophotometer at six analytical wavelengths in the range of 450-650 nm. Next, the scattering efficiency factor (p) and the scattering coefficient K (p) are determined, and the hemoglobin concentration in the blood is calculated by the formula: HB = 128 · D ₈₀₅ · p / K (p), where HB is the concentration of hemoglobin in the blood; D ₈₀₅ - optical density of the suspension at 805 nm; p is the scattering efficiency factor; K (p) is the scattering coefficient. In addition, for the method [patent RU No. 2140083, G01N 033/52 G01N 033/72] the measured optical densities judge the concentration of the main hemoglobin derivatives.

The disadvantages of this method include the technical complexity of the procedure associated with the measurement of cocking red blood cells at three and six wavelengths, which increases the total time for one analysis. Also, a significant drawback is the removal of two measuring indicators of light scattering from a spectrophotometer, which, on the one hand, increases the calculation operations, which leads to an increase in the measurement errors of hemoglobin concentration, and, on the other hand, increases time expenditures. In addition, the maximum absorption of the hemoglobin solution is at a wavelength of 540 nm, which for the method [patent RU No. 2140083, G01N 033/52 G01N 033/72] is not fulfilled. The latter reduces the sensitivity of the proposed method, which limits its use in disaster medicine, sports physiology, and veterinary medicine.

Also known is a method for determining the concentration of hemoglobin in blood samples [US patent No. 2062465, G01N 033/48] by placing a sample of whole blood in a capillary tube with a plastic float. The concentration of hemoglobin in the red blood cells compacted after centrifugation is measured by determining the depth at which the float is immersed in the layer of red cells, and then its concentration in the blood is calculated. The concentration of hemoglobin in whole blood is calculated by multiplying the average corpuscular concentration of hemoglobin in the packed red blood cells by the volume percentage of packed cells (hematocrit) in whole blood.

The disadvantages of this method include a high error in measuring the concentration of hemoglobin of the proposed method. Since the percentage of condensed cells (hematocrit) in whole blood also depends on the patient’s functional state (sick or healthy, after sleep or wakefulness, an excited or suppressed emotional state, pregnancy, etc.), and not only on the hemoglobin content in red blood cells, then we can assume a low accuracy in determining the concentration of hemoglobin in blood samples. In addition, the proposed method depends on the volume of the analyzed blood and blood dilution, which also reduces the accuracy of the measurement procedures.

Closest to the claimed method is a method for determining hemoglobin in blood and urine samples using a sensitive reaction to chlorpromazine [Chalmers A.H., Lesley E.S. Evaluation of hemoglobin in plasma and urine by spectrophotometric method / article /. Clinical Chemistry, Vol.39, No.8, 1993, p.1679-1682] by adding the following reagents to the control and experimental cuvette: 1. Acid mixture (KS): concentrated acetic acid - 28 ml; concentrated phosphoric acid - 4 ml .; 2. A solution of egg albumin (YA): YA - 200 mg; distilled water - 10 ml (shelf life: 6 hours at t = 4 ° C); 3. A solution of chlorpromazine: chlorpromazine - 250 mg; distilled water - 25 ml (shelf life: 8 days at t = 4 ° C); 4. Hydrogen peroxide: hydrogen peroxide (30%) - 1 ml; distilled water - 9 ml (shelf life: 6 hours at t = 4 ° C). Then, undiluted plasma or urine is added to the experimental cuvette of the spectrophotometer in a volume of 20 μl and photometric at a wavelength of 528 nm and the increase in optical density against the control sample is determined. Moreover, for each determination, three parallel measurements of optical density are carried out with the subsequent determination of the average value.

The disadvantages of this method include:

1) the duration of the procedure (10-15 minutes per analysis);

2) the technical complexity in the execution, which consists in preparing, to increase the accuracy of measurements, three parallel samples of a suspension of red blood cells, which leads to the duration of one analysis and reduces the accuracy of determining the concentration of hemoglobin;

3) the toxicity of the reagents used (chlorpromazine, concentrated acids);

4) low safety of the quality of the applied reagents (not more than one week from the date of their preparation);

5) low sensitivity to the trace presence of hemoglobin in biological fluids (blood, urine, saliva).

The main objectives of the invention are: increasing the specificity and sensitivity of the method for determining the concentration of hemoglobin (Hb), simplifying the technical execution procedure, reducing the consumption of reagents and the degree of toxicity of the reagents used.

This is achieved due to the fact that in the method for determining hemoglobin in biological fluids by photometry of their samples, which involves introducing a mixture of two concentrated acids into the control and experimental cuvettes, one of which is acetic, adding egg albumin solution, introducing a biological fluid sample into the experimental cuvette and solution chlorpromazine in both cuvettes, adding a solution of hydrogen peroxide and registering the increase in optical density of the solution in the experimental cuvette against the density in the control cuvette, suggests As the second concentrated acid, use nitric acid with an acid ratio in the mixture 1: 1 by volume, add egg albumin solution and hydrogen peroxide solution only to the experimental cuvette, inject chlorpromazine solution at a concentration of 2.5 mg per 100 ml of distilled water and record the optical density at a wavelength in the range of 530-550 nm.

As a biological fluid, a sample of whole blood or its dry sample or saliva is injected in a volume of 5 μl to trace amounts, while a sample of whole blood is previously diluted 2000 times.

Use photometric cuvettes with a thickness of 2.5 mm with inserts made of glass or plexiglass to reduce their volume.

For photometry, a photoelectrocolorimeter is used.

And also reveal a linear relationship between the concentration of hemoglobin and the increase in the optical density of the solution in the experimental cell for different concentrations and calculate the regression equation to calculate the concentration of hemoglobin in the solution:

Hb (mg / L) = - 9.84+ (2409.94 × ΔD),

where ΔD is the difference in optical density between the experimental sample and the control.

The development of the proposed method is aimed at minimizing the input reagents, which allows to increase the accuracy and expressness of the definitions.

The decrease in the concentration of chlorpromazine solution from 250 mg per 25 ml of distillate to 2.5 mg per 100 ml of distilled water significantly reduces the consumption of reagents and, unlike the prototype, does not require three parallel measurements, thereby significantly simplifying the measurement procedure and increasing the accuracy of determining the concentration hemoglobin.

A mixture of concentrated acids in the indicated volumes creates a stable buffer solution with an experimental mini-test with a longer maintenance of the pH value of the photometric liquid at the physiological level, in comparison with the prototype.

For analysis, a wavelength in the range of 530-550 nm was used, which increases the accuracy of determining the hemoglobin content in blood samples, since the maximum absorption of a hemoglobin solution is in this range [Kozinets GI, Arustamyan A.Yu., Ashurov G.D . Blood system studies in clinical practice. - M .: Triad X, 1997. - 480 p.].

A standard Hb solution with egg albumin solution was added to the test cuvette to prevent adhesion of the standard Hb solution to the walls of the tube and cuvette.

Distilled water (as a cheaper and more affordable material) was added to the control cuvette instead of egg albumin solution.

The volume of diluted whole blood and other biological fluids is necessary in an amount of 5 μl or less (up to traces of presence and dry samples), which can be used in forensics.

Using a photoelectric colorimeter allows you to achieve several advantages: its small size makes it more transportable, which is necessary in some cases, it does not require qualified personnel during operation, it is less expensive compared to the stationary spectrophotometer used in the prototype.

The method for determining hemoglobin is as follows: 0.8 ml of an acid mixture (a mixture of equal volumes of concentrated acetic and nitric acids) is added to a control and experimental photometric cuvette with a thickness of 2.5 mm and plexiglass inserts. A solution of egg albumin in distilled water in a volume of 10 μl and 5 μl of biological fluid (whole blood diluted 2,000 times, or its dry sample, urine, saliva) and 0.25 ml of chlorpromazine solution (with a concentration of 0.0025 %), which is introduced in the same volume into the control cell. Then, in order to start the reaction of chlorpromazine with heme-containing compounds, 0.5 ml of three-percent hydrogen peroxide is added to the experimental cuvette and strictly after 2-3 minutes the increase in the optical density of the solution is recorded at a wavelength of 530-550 nm against the control sample.

A linear relationship is found between the concentration of hemoglobin (Hb) and the increase in optical density in the solution, which made it possible to calculate the regression equation to calculate the concentration of Hb in the solution:

where ΔD is the difference in optical density between the experimental sample and the control.

To convert the hemoglobin concentration from mg / l to the g / l used in medicine and taking into account the dilution, a recalculation formula is proposed:

For routine photometric determinations, a KFK-3 photoelectrocolorimeter was used. To derive the regression equation, a standard Hb solution of different concentrations was used. Based on these data, a calibration graph for whole blood (Fig. 1) and a calibration graph for urine and saliva (Fig. 2) were constructed, and a high correlation coefficient (r) of 0.984 was obtained, which allowed us to consider the proposed modification as valid. Due to the high sensitivity of the chlorpromazine method according to the calibration graph, Hb is measured in mg / l in diluted blood. Accordingly, the concentration of Hb is determined from this graph or from formulas (1) and (2).

Thus, the proposed method based on the use of chlorpromazine mini-method has:

1) specificity and high sensitivity, that is, it responds exclusively to heme-containing (hemoglobin) compounds and is able to determine the quantitative content of hemoglobin in microscopic doses (traces);

2) expressity (from 10-15 minutes to 2-3 minutes per analysis);

3) technical simplicity in execution;

4) economical reagent consumption;

5) a reduced degree of toxicity of the reagents by preparing a more dilute solution of chlorpromazine;

6) longer storage of diluted chlorpromazine solution (from one week to several months);

7) high sensitivity and accuracy to the trace presence of hemoglobin in biological fluids (dry blood, urine, saliva).

Example 1. The accuracy of the chlorpromazine micro-method was controlled by reproducible parallel analysis of blood samples of 34 individuals being treated in the burn center of Kemerovo and the KemSU dispensary using hemiglobin cyanide modified chlorpromazine methods and an analyzer calculation method. Statistical indicators of the variation in the results are shown in Table 1. It can be seen that the chlorpromazine method reflects the hemoglobin content in the different studied groups most accurately and with a smaller variation spread. The increased level of Hb in burn patients can be explained by an increase in blood viscosity during a burn, to which the body responds by increasing the amount of Hb in the blood.

Example 2. Example 2 shown in table 1 is carried out analogously to example 1, only 10 blue pigeons of the city of Kemerovo and 7 pigeons of the city of Mariinsk were taken as the examined groups. It is seen that the concentration of hemoglobin in pigeons is higher than in humans. This can be explained by the presence of nuclei in the erythrocytes of pigeons, which greatly influenced the optical density of the solution. The high concentration of Hb in pigeons of the Kemerovo city is explained by unfavorable environmental conditions.

Example 3. The sensitivity of the chlorpromazine method was tested using the recommendations of V. V. Menshikov (Laboratory research methods in the clinic: Handbook. - M., 1987) by finding a concentration of the test substance (in this case, hemoglobin) that would correspond to the smallest measurement result , still different from the value of the blank sample (see figure 2). For this, in five parallel determinations we measure the optical density of a blank sample, which corresponds to a certain average concentration of hemoglobin. Further, according to V.V. Menshikov, the average value of the hemoglobin concentration is substituted into the formula: x + 3S, where x is the average value of the blank sample, S is the standard deviation, we obtain a value that should approximately correspond to the minimum value of Hb in urine and saliva ( see figure 2), since in the hemolysate of whole blood a deliberately higher level of hemoglobin. Indeed, in Table 2 we find that the minimum Hb concentration (in g / l) of the second sample fits into the value of the blank sample, which indicates a high sensitivity of the method to the trace presence of the substance. Thus, the sensitivity of the method is 0.5 g / l, while the sensitivity of the hemichromic method is 25 g / l.

Example 4. On the same samples of whole blood, the quantitative characteristics of which are shown in table 1, the sensitivity of the chlorpromazine method was tested (table 2). The same samples of whole blood were diluted 100 times and received hemoglobin concentrations correspondingly 100 times lower than those shown in table 1 and shown in brackets in table 2. In all samples of whole blood, the chlorpromazine method yielded slightly overestimated results, whereas the unified hemiglobin cyanide method gave a negative reaction in all cases (staining of the solution was not observed), that is, did not detect the presence of traces of hemoglobin in the studied samples. Based on the results obtained, we can state the high sensitivity of the chlorpromazine method.

Table 1. Comparison of hemoglobin determination results (M ± m) by three independent methods Examples Surveyed groups (n) Methods for the determination of hemoglobin, g / l Calculation method Hemiglobin cyanide Chlorpromazine one Healthy people, n = 14 138 ± 8 127 ± 16 141 ± 6 Burn patients, n = 20 134 ± 7 141 ± 10 156 ± 4 2 Pigeons, n = 10 (Kemerovo city) 132 ± 7 157 ± 9 176 ± 5 Pigeons, n = 7 (Mariinsk) 130 ± 9 156 ± 9 172 ± 6 Table 2. Sensitivity testing of the chlorpromazine method (M ± m) Sample Name The optical density (D,%) Hb concentration in mg / l Hb concentration in g / l Idle test 9 ± 0.15 0.89 ± 0.06 0.56 ± 0.02 The sample corresponding to the minimum optical density of the measurement of Hb in urine and saliva (see figure 2) 14 ± 0.2 1.09 ± 0.02 0.57 ± 0.01 Healthy people, n = 14 - 2.42 ± 0.07
(1.41 ± 0.06) 1.271 ± 0.07
(1,686 ± 0,06) Burn patients, n = 20 - 3.12 ± 0.05
(2,971 ± 0,04) 1,638 ± 0,05
(1.56 ± 0.04) Pigeons, n = 10 (Kemerovo city) - 3.67 ± 0.06
(3.352 ± 0.05) 1.923 ± 0.06
(1.76 ± 0.05) Pigeons, n = 7 (Mariinsk) - 3.42 ± 0.07
(3.276 ± 0.06) 1.80 ± 0.07
(1.72 ± 0.06)

Claims (5)

1. A method for determining hemoglobin in biological fluids by photometric measurement of their samples, comprising introducing into the control and experimental cuvette a mixture of two concentrated acids, one of which is acetic, adding an egg albumin solution, introducing a biological fluid sample into the experimental cuvette and chlorpromazine solution in both cuvettes, adding hydrogen peroxide solution, and registration of the increase in optical density of the solution in the experimental cuvette against the density in the control cuvette, characterized in that as the second end nitric acid is used with nitric acid at a ratio of acids in the mixture 1: 1 by volume, egg albumin solution and hydrogen peroxide solution are added only to the test cuvette, chlorpromazine solution is introduced at a concentration of 2.5 mg per 100 ml of distilled water and the absorbance is recorded at a wavelength in the range of 530-550 nm. 2. The method according to claim 1, characterized in that as a biological fluid, a sample of whole blood or its dry sample, urine or saliva is injected in a volume of 5 μl to trace amounts, while the whole blood sample is pre-diluted 2000 times. 3. The method according to claim 1, characterized in that for placing a sample of biological fluid using photometric cuvettes with a thickness of 2.5 mm with inserts made of glass or plexiglass. 4. The method according to claim 1, characterized in that to determine the optical density using a photoelectric colorimeter. 5. The method according to one of claim 1 or 7, characterized in that a linear relationship between the concentration of hemoglobin and the increase in optical density of the solution in the experimental cell for different concentrations is detected and the regression equation is calculated to calculate the concentration of hemoglobin in the solution Hb (mg / L) = - 9.84+ (2409.94 · ΔD), where ΔD is the difference in optical density between the experimental sample and the control.

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