RU2497456C1 - Method of express-diagnostics of poisoning with hemolytic poisons - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, namely to toxicology and clinical laboratory diagnostics. In order to carry out express-diagnostics of poisoning with hemolytic poisons, analysis of plasma of peripheral, including capillary, blood is performed. With application of 0.005-0.01% solution of sodium dodecylsulfate in blood plasma sample all hemoglobin fractions are transformed for several seconds with ratio sample:reagent 1:10. After that, sample is photometrically scanned at wavelengths 540 and 700 nm, and concentration of ectoglobular hemoglobin is calculated. If value of ectoglobular hemoglobin is higher than 1.0 g/l, poisoning with hemolytic poisons is diagnosed.

EFFECT: method eliminates poisonous component from reagent composition, reduces time of analysis duration, reduces interference of sample components, minimises quantity of biomaterial required for test, increases diagnostics efficiency.

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Description

The present invention relates to medicine, namely to toxicology, clinical laboratory diagnostics, and can be used for rapid diagnosis of hemolytic poisoning poisoning both in hospitals laboratories and in the field.

The problem of acute and chronic poisoning has acquired particular urgency in recent decades due to the accumulation in the environment of a huge amount of chemicals of anthropogenic origin. According to the World Health Organization, in the 60s. in Western Europe, on average, 1 person per 1000 inhabitants was hospitalized for acute poisoning, in the 90s. - almost twice as much. These patients account for up to 15-20% of all persons urgently admitted to hospital treatment for urgent reasons. In total, in the world in 2000, about 250,000 people died from poisoning - 4.3% of the total number of deaths (Luzhnikov E.A., Sukhodolova G.N., 2008). Among household poisoning and suicidal attempts, the leading place is taken by medicines, acetic acid and technical fluids. In acute poisoning, blood damage is usually caused by toxic effects of hemotoxicants on red blood cells with the formation of pathological forms of hemoglobin or hemolysis, which causes hemic hypoxia. Quick and affordable methods for the diagnosis of chemical poisoning are of particular relevance for clinical and military toxicology (Sokolova N.A., Vitkovsky Yu.A., Govorin A.V., 2011; Kiryushin V.A. et al., 2004).

As a rule, the diagnosis of hemolytic poisoning is established on the basis of laboratory determination of the concentration of extra-erythrocytic hemoglobin (VEG) that appears in blood plasma as a result of direct toxic damage to the membranes of red blood cells (Luzhnikov E.A., Sukhodolova G.N., 2008).

A known method for determining the concentration of VEG, as an additional diagnostic method. The study was carried out using the hemiglobin cyanide method at a wavelength of 540 nm (Levina L.D., Bezuglova I.A., Bezuglov D.A., 1993). To do this, blood in an amount of 2-3 ml was taken from a vein with a dry cold needle, “gravity” collecting in a dry test tube. After centrifugation (10 minutes at 1500 rpm), 0.4 ml of test serum was added to the cuvette of the SF-46 spectrophotometer with an optical path length of 10 mm. Immediately before spectrophotometry, 1.6 ml of the standard transforming reagent Drabkin was added. The optical density was determined at a wavelength of 540 nm against the transforming solution. To calculate the concentration of VEG used the formula VEG = E _{540 *} 16.1 _* 5 / 11.0, where E ₅₄₀ is the extinction of the test solution; 16.1 - relative molecular weight of hemoglobin; 5 - serum dilution factor; 11.0 - millimolar extinction coefficient for hemiglobin cyanide.

The disadvantages of the method are: the use of serum for analysis, which increases the study time by 20-30 minutes due to blood coagulation, a large volume of serum (0.4 ml), toxicity of the reagent (potassium cyanide), and interference of compounds absorbing at this wavelength ( bilirubin, triglycerides). A study not on an empty stomach or in patients with jaundice leads to false positive results and overdiagnosis. In addition, the authors did not determine the concentration discriminant above which intravascular hemolysis is diagnosed.

A known method adopted for the closest analogue of determining the concentration of VEG of blood plasma (serum) by the hemiglobin cyanide method (Tonkoshkurova O.A., Dmitriev A.I., Dmitrieva R.E., 1996), based on the above reaction with the same reagent, characterized photometry at an additional wavelength of 670 nm. The method was carried out as follows: to 0.5 ml of serum (plasma) blood was added 3 ml of distilled water, 0.5 ml of a transforming solution, stirred and after 1-2 minutes the optical density was fixed at a wavelength of 540 nm in a cuvette with an optical path length of 10 mm) against water as a blank test. Then, without removing the cuvette from the photoelectrocolorimeter, the optical density was recorded at a wavelength of 670 nm. The VEG concentration was calculated by the formula: VEG (g / l) = (D540-D670) × K × 8/251, where D540 and D670 are the optical density values measured at 540 and 670 nm; K is the calibration coefficient found in accordance with the instructions for the standard set of reagents for determining hemoglobin by the hemiglobin cyanide method; 8 - the degree of dilution of serum (plasma); 251 - blood dilution recommended by the instructions for the reagent kit.

The disadvantages of the method are: a large sample volume (0.5 ml), the toxicity of the reagent (potassium cyanide) and the absence of a specific value, above which hemolysis is diagnosed.

Tasks: elimination of the toxic component from the reagent, reducing the analysis time, reducing the interference of the sample components, minimizing the amount of biomaterial needed for the study, increasing the efficiency of diagnosis.

The inventive method for the rapid diagnosis of hemolytic poisoning poisoning, including a study of blood plasma, characterized in that when using a 0.005-0.01% solution of sodium dodecyl sulfate in a peripheral plasma sample, including capillary blood, all hemoglobin fractions are transformed within a few seconds when the ratio of sample: reagent is 1:10, photometry at wavelengths of 540 and 700 nm, the concentration of extra-erythrocyte hemoglobin is calculated, and when the value of extra-erythrocyte hemoglobin is greater than 1.0 g / l, they are diagnosed with The pressure hemolytic poisons.

The technical result of the invention is to improve the quality and timeliness of the diagnosis of poisoning with hemolytic poisons by reducing the interference of bilirubin and triglycerides in the sample, the possibility of using capillary blood instead of venous plasma for analysis, reducing the amount of material needed for analysis by 5-10 times, and reducing the study time by 4 -5 times, minimizing staff labor, using a non-toxic reagent without cyanide salts. All this in general contributes to an increase in the level of toxicological assistance and a reduction in mortality in hospitalized patients.

The method is as follows.

Blood is taken from 0.2-0.5 ml from a patient from any accessible peripheral vessel (artery, vein) or from the capillary bed (distal phalanx of the finger or earlobe). Blood in a test tube is mixed with a drop of a solution of any anticoagulant to prevent clotting and plasma production. The type of anticoagulant does not affect the result of the study, so it can be selected based on the capabilities of the laboratory and hospital (sodium heparin, lithium heparin, ethylene diamine tetraacetate trikali, sodium citrate). Immediately after collection, the blood sample is centrifuged at 1500-3000 rpm for 2-5 minutes.

0.1 ml of plasma is mixed with 1.0 ml of a 0.005-0.01% sodium dodecyl sulfate solution, shaken for several seconds, poured into a photometer cuvette and photographed twice at wavelengths of 540 and 700 nm, respectively. The difference in optical densities is calculated by the formula: E _Hb = E ₅₄₀ -E ₇₀₀ .

Calculation of the concentration of VEG in the sample is carried out either by multiplying the difference in optical densities by the molar extinction coefficient (K = 10.16), or according to a calibration graph constructed using standard hemoglobin solutions - hemolysates (Dolgov V.V., Ovanesov E.N., Schetnikovich K.A., 2004). With a VEG value of more than 1.0 g / l, poisoning with hemolytic poisons is diagnosed.

The proposed method was tested on 162 simulated samples of human blood with various anticoagulants, the osmotic hemolysis of which was caused by the addition of distilled water in a ratio of 10: 1, respectively. After an hour-long exposure, the VEG concentration was determined in them by the hemiglobin cyanide and the proposed method, as well as total Endrashik-Grof bilirubin and triglycerides by the enzymatic photometric method. Comparative characteristics of the methods for the determination of VEG are given in table 1.

Table 1 Comparative characteristics of methods for the determination of extraerythritic hemoglobin. The closest analogue The proposed method Incubation time 1-2 minutes 15-30 seconds Sample volume 0.5 ml 0.02-0.1 ml Reagent volume 3.5 ml 0.2-1.0 ml Sample / Reagent Ratio 1/7 1/10 The presence of cyanides in the composition of the reagent Yes No

As a result of the experimental part of the work, the analytical characteristics of the proposed method were determined:

- sensitivity - 0.02 g / l,

- linearity - up to 28.00 g / l,

- coefficient of variation - 7.0%.

When VEG 613 concentration study in healthy volunteers following results were obtained: median - 0.16 g / l, 25 ^th and 75 ^th percentile - 0.09 and 0.19 g / l respectively.

The results of the correlation analysis of the prototype and the proposed method are given in the appendix “Correlation between the concentration of VEG determined in two ways”. The graph shows a strong positive correlation between the two methodological approaches (r = 0.878 at p << 0.0001), which indicates the analytical qualities of the method no worse than the prototype (see drawing).

To assess the interference of total bilirubin (C _beat ) and triglycerides (C _trig ) using both methods, a regression analysis was also applied for the VEG and the so-called “interference value” (SI) calculated as C _{beat *} C _{trig *} 5. Coefficient the correlation for IV and the prototype method was 0.808, for VI and the proposed method 0.740, which indicates less interference of bilirubin and triglycerides in determining the VEG of the proposed method.

In addition, we tested the automation of the proposed method for performing on biochemical analyzers of various capacities (A-25, BioSystems, Spain; AU-640, Olympus, Japan; KoneLab-20, ThermoScientific, Finland). An example is the application of the methodology (table 2), reflecting the proposed method, for the automatic biochemical analyzer "A-25", BioSystems (Spain).

table 2 An example of the application of the proposed method for determining non-erythrocyte hemoglobin for programming automatic biochemical analyzers. Nonerythrocyte Hemoglobin Serum / plasma Common parameters: Analysis mode Monoreactive. End point Turbidimetric test No Units g / l Reaction type Increasing Number of repetitions one Decimal places 2 The name of the test in the report for the patient VEG Procedure: Reading Bichromatic Main wavelength 540 nm Reference wavelength 700 nm Sample volume 20 μl Reagent volume 1 200 μl Reagent volume 2 - Flushing volume 1.0 ml Read Time 1 Cycles - 3 / Seconds - 36 Read Time 2 - Reagent addition time 2 - Pre-breeding factor - Post-breeding factor - Auto repeat No Calibration: Repeat Form 2 Calibrator Repeat 3 Calibration type Monocalibrator Factor - Concentration 0.6 Calibration curve Linear Controls: Number of controls 2 Repeat control one Sigma 3 Type of control Water. Mono. Options: Reagent only form No Saline form Yes Blank Absorption Limit 0.05 Kinetics form limit - Linearity limit 28.00 g / l Sensitivity limit 0.02 g / l Factor limits - Normal values 0.00-0.30 Repeat Categories -

With appropriate laboratory capabilities, the automation of the method not only facilitates and increases the reliability of diagnosis, reducing the labor costs of medical personnel, but also significantly reduces the amount of material needed for analysis (in our example, only 0.02 ml of plasma).

The clinical part of the testing was carried out on 19 patients with a verified diagnosis of acetic acid poisoning, as the most common type of poisoning with hemolytic poisons in everyday life, hospitalized in the Department of Anesthesiology and Resuscitation No. 7 of the Regional Clinical Hospital No. 1 of Krasnodar in 2005-2011.

Example 1. Patient K., 28 years old (IB 2039). I entered the Department of Anesthesiology and Resuscitation No. 7 of the Regional Clinical Hospital No. 1 of Krasnodar on 02/04/2009 with a diagnosis of poisoning with vinegar essence.

Upon admission, an extremely serious condition of the patient was noted. In addition to standard laboratory tests, on the first and third days, the concentration of VEG was determined by the proposed method: 0.5 ml blood was taken from a cubital vein patient, which was mixed in a test tube with a drop of sodium hepinate solution and centrifuged for 3 minutes at 2500 rpm .

0.1 ml of plasma was mixed with 1.0 ml of a 0.005% sodium dodecyl sulfate solution, shaken for several seconds, and photographed twice at wavelengths of 540 and 700 nm, respectively. The difference in optical densities was calculated by the formula: E _Hb = E ₅₄₀ -E ₇₀₀ . According to the calibration schedule, the concentration of VEG was determined - 29.61 g / l.

The concentration of VEG exceeded 1.0 g / l by almost 30 times. The patient was confirmed poisoning with hemolytic poisons (acetic acid), intensive therapy with the use of efferent methods was immediately started, on the third day there was a positive dynamics of red blood counts (table 3).

Table 3 Dynamics of the results of laboratory studies of patient K., 28 years old. Tests 1 day 3 days Hemoglobin of whole blood, g / l 92 103 Red blood cells, × 10 ¹² / l 2.03 3.03 VEG, g / l (proposed method) 29.61 10.92

After 21 days, the patient was transferred to the gastroenterological department for aftercare. Later in the ENT center, esophagus was bougiended to restore its patency. In satisfactory condition, the patient was discharged home.

Example 2. Patient L., 34 years old (IB 39113). Was admitted to the anesthesiology and intensive care unit No. 7 of the Regional Clinical Hospital No. 1 of Krasnodar on 08/08/2009 with a diagnosis of alcohol intoxication, poisoning with acetic acid?

Upon admission due to alcohol intoxication and the absence of relatives, a medical history was not possible. The patient's condition was stable: no pronounced hemodynamic disturbances were observed, there was no blood in the gastric discharge, the diuresis was preserved. In addition to standard laboratory tests at admission, VEG concentration was determined by the proposed method: 0.5 ml of blood was taken from a cubital vein of a patient, which was mixed in a test tube with a drop of tripotassium ethylene diamine tetraacetate solution and centrifuged for 5 minutes at 1500 rpm.

0.1 ml of plasma was mixed with 1.0 ml of a 0.01% sodium dodecyl sulfate solution, shaken for several seconds, and photographed twice at wavelengths of 540 and 700 nm, respectively. The difference in optical densities was calculated by the formula: E _Hb = E ₅₄₀ -E ₇₀₀ . According to the calibration schedule determined the concentration of VEG - 0.21 g / L.

According to the proposed method, the concentration of VEG did not exceed 1.0 g / l, which eliminates poisoning with hemolytic poisons. Other indicators of red blood were also within normal limits (table 4).

Table 4 The results of laboratory tests patient L., 34 years old, upon admission to the hospital. Tests Result Hemoglobin of whole blood, g / l 123 Red blood cells, × 10 ¹² / l 3.96 VEG, g / l (proposed method) 0.21

After stopping alcohol intoxication, the patient admitted that there was no fact of taking acetic acid. In connection with the normalization of the general condition, the patient was transferred to the gastroenterological department and was subsequently discharged.

Conclusions: the proposed method allows to improve the quality and timeliness of the diagnosis of poisoning with hemolytic poisons by reducing the interference of bilirubin and triglycerides, the possibility of using capillary blood instead of venous for analysis, reducing the amount of material needed for analysis by 5-10 times, reducing the study time by 4-5 times, use of a non-toxic reagent without cyanide salts. All this, in general, helps to increase the level of toxicological assistance and reduce mortality in case of poisoning with hemolytic poisons.

Information sources

1. Luzhnikov EA, Sukhodolova G.N. Clinical toxicology. - 4th ed., Revised., And add. - M .: Medical Information Agency LLC, 2008. - 578 p.

2. Sokolova N.A., Vitkovsky Yu.A., Govorin A.V. The defeat of the cardiovascular system in acute poisoning with acetic acid. - Tomsk; Chita: Ivan Fedorov Publishing House, 2011. - 120 p.

3. Kiryushin V.A. et al. Toxicology of chemically hazardous substances and activities in foci of chemical damage. - Ryazan, 2004 .-- 163 p.

4. Levina L.D., Bezuglova I.A., Bezuglov D.A. The clinical significance of the study of free hemoglobin in blood serum in viral hepatitis // Laboratory work. - 1993. - No. 3. - S.23-25.

5. Tonkoshkurova OA, Dmitriev A.I., Dmitrieva R.E. Determination of the concentration of non-erythrocyte plasma hemoglobin (serum) by the hemiglobin cyanide method // Clinical Laboratory Diagnostics. - 1996. - No. 2. - S.21-22.

6. Dolgov V.V., Ovanesov E.N., Shchetnikovach K.A. Photometry in laboratory practice. - SPb, 2004. - S.66-79.

Claims (1)

A method for the rapid diagnosis of hemolytic poisoning poisoning, including a study of blood plasma, characterized in that when using a 0.005-0.01% sodium dodecyl sulfate solution in a peripheral plasma sample, including capillary blood, all hemoglobin fractions are transformed for several seconds at a sample ratio : reagent 1:10, photometry at wavelengths of 540 and 700 nm, the concentration of extra-erythrocyte hemoglobin is calculated, and if the value of extra-erythrocyte hemoglobin is more than 1.0 g / l, hemolytic poisoning is diagnosed E poisons.

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