The method for determining the acid resistance of erythrocytes
The invention relates to medicine, namely to laboratory diagnosis. To evaluate the acid resistance of erythrocytes erythrocyte suspension will standardize the breeding of erythrocyte mass saline solution in a ratio of 1:3 and centrifugation for 15 min, followed by dilution of 0.01 ml of erythrocyte sediment 4.0 ml of physiological solution. Add acid hemolytic, which is used as a 1% solution of acetic acid. Determine the amount of light transmission through the parallel account changes in the average size of red blood cells and the amount of light transmission with graphing, which determine the time of registration the maximum average size of erythrocytes and the average rate of hemolysis. Calculate the index of acid resistance of erythrocytes (X) by the formula: X= t_d max/gVm, where t_d max - time maximum average radius of erythrocytes, sec; gVm - average rate of hemolysis, the river. units, the value of X from 0.50 to 0.68 consider normal. The application of the method allows to estimate the parameters of the rigidity and durability of erythrocyte pool necessary for the diagnosis of anemia of different origin. 1 Il.
This purpose is often used methods to evaluate the resistance of erythrocytes to the influence of various factors that may cause their destruction, that is, to cause hemolysis.
There is a method of determining the osmotic resistance of erythrocytes Limbuko and Ribiero (Manual of clinical laboratory research, Medgiz, 1960, pp. 96-98), in which the use of hypotonic solutions with salt concentrations from 0.56 to 0.28%. They poured in a row of test tubes, then the same pipette from geometry Sali in each tube is poured 0.02 ml of blood taken from the pulp of the finger. Careful shaking of the tubes reach a uniform mist of blood in saline solution. The tubes leave the tripod at room temperature for 1 hour, then within 5 minutes, centrifuged at 2000 rpm, after which mark the maximum and minimum level of osmotic resistance.
The reading of the results.
The maximum and minimum level of resistance is the salt concentration at which hemolysis began and ended.
The maximum level of resistance, that is, the first signs of hemolysis determined by eye light yellow, and the level of minimum durability (complete destruction of erythrocytes) - intensively to the school resistance fluctuations in the range of 0.48-0.46 per cent; for a maximum of 0.34-0.32 per cent.
Lowering the resistance of red blood cells, according to the authors, the method has a place in congenital spherocytosis, and acute hemolytic crisis and vitamin B12 deficiency anemia expanding the boundaries of resistance.
The disadvantage of this method is its inaccuracy related to the fact that
1) results are taken into account through 3-4-24 hours, whereas osmotic hemolysis of erythrocytes is performed rapidly, reaching its maximum after 5 minutes;
2) increased hemolysis long periods of time may occur due to microbial growth and due to the absorption of carbon dioxide in the air;
3) inaccurate visual assessment as the minimum and maximum hemolysis.
The closest achieved a positive result is a method of evaluating the resistance of erythrocytes to acid hemolytica by determining the amount of light transmission of standardized suspension of erythrocytes with graphing hemolysis (Laboratory work. - 1965. No. 9. - P. 530-531).
The course definition.
Taken from a finger puncture 1-2 drops of blood and placed in a tube containing 2-3 ml of saline.
Standardization of conditions is achieved by bringing the optical density of the suspension of erythrocytes to the greates what Amnesty 0,7 D and placed in the cuvette in the right light beam fotoelektrokalorimetry. In the left slot insert a cuvette containing saline solution.
To the suspension of erythrocytes add 2.0 ml of 0.004 N hydrochloric acid and immediately noted the testimony of FEC on the right reel. Then take readings every 30 seconds as long as they do not become permanent. The value of optical density is written in the column opposite corresponding to the timestamp.
To plot the acid resistance of erythrocytes produce the following calculations.
From the first readings of optical density subtract the latter. So recognize the change in optical density, corresponding to 100% hemolysis. The obtained result is divided by 100 and get the change in optical density per 1% of hemolysis.
1. From each of the previous readings of optical density is deducted from a subsequent and receive optical density in each subsequent 30 seconds.
2. Data obtained under paragraph 2, is divided by the optical density corresponding to 1% of hemolysis (item 1), and receive a percentage hemosiderosis erythrocytes for each subsequent 30 seconds.
3. The results obtained are expressed graphically (see drawing).
Possible avtra EFA-1.
The course definition.
In the cuvette is placed in 2.0 ml of the suspension of erythrocytes and 2.0 ml of 0.004 N hydrochloric acid (stylus should be rejected in its rightmost position) and include the recording mechanism. The record is produced on a paper tape with a millimeter grid. With the development of hemolysis recorder trace along the integral curve of optical density of the suspension of erythrocytes.
Processing of the material.
The resulting curve is enclosed in a coordinate system so that the zero mark coincides with the beginning of the recording, the abscissa shows the delay time of hemolysis, and the ordinate axis is the optical density.
The change in optical density corresponding to the hemolysis for each subsequent 30 seconds, easy to find, comparing the segments on the axes of abscissa and ordinate.
The percentage of broken red blood cells per 1 mm of the scale of the ordinate, is found by measuring in millimeters the total change in optical density, which is taken for 100%. Erythrocytes can be divided into the following groups: decreased-persistent - broken for the first third of the entire time of hemolysis, rednisone for the second third and value-resistant, broken for the last third of the time of hemolysis.
In addition, you can calculate the total from the cylinder the appropriate time.
This method, as well as similar, is used for the diagnosis of various anemias.
The disadvantages of this method are:
1) complexity; (2) duration; 3) the inability to assess the dynamics of the elasticity of erythrocytes under the influence of acid hemolytica.
A positive result of the proposed method is the determination of the parameters of the elasticity of red blood cells to fracture and increase the transmittance of the suspension of erythrocytes under the influence of acid hemolytica for a more accurate assessment of resistance of erythrocytes to the effects of acid hemolytica largest index of acid resistance.
A positive result is achieved by the fact that the resulting standardized level of dilution of red blood cells allows to construct curves of hemolysis and the elasticity of red blood cells using analyzer aggregation “Biola”, which is used in many clinical laboratories in Russia only for the evaluation of platelet aggregation, and to calculate an index of acid resistance, decreasing or increasing in the presence of the investigated blood of a large number of red blood cells, prone, or more resistant to destruction by acid hemolytica.
The method is performed as education is ifoga laboratory (ARF-U,2).
1) the anticoagulant of 3.8% solution of sodium citrate (produced by dissolving 3.8 g of sodium citrate (REACHIM OFS-6-4 Mikhailovsky plant chemicals) in 100 ml of distilled water);
2) a 0.85% solution of sodium chloride (produced by dissolving 0.85 grams of sodium chloride (REACHIM OFS-6-4 Mikhailovsky plant chemicals) in 100 ml of distilled water);
3) acid hemolytic - 1% solution of acetic acid (receive, adding to 10.2 ml of 98% acetic acid in 89.8 ml of distilled water).
The course definition.
From the cubital vein of a wide dry needle, blood is taken in a volumetric flask with 3.8% sodium citrate in a ratio of 9:1. A slight rocking or shaking the blood mixed with anticoagulant.
The blood is centrifuged in a centrifuge at 1500 rpm for 15 min, after which the plasma is sucked off by pipette. Received RBC mass bred a 0.85% solution of sodium chloride in the ratio of 1:3 and centrifuged 15 min at 1500 rpm After centrifugation the supernatant is sucked off by pipette, and the remaining RBC mass is thoroughly mixed to a homogeneous state. In a test tube with 4 ml of 0.85% sodium chloride solution, add 0.01 ml of the obtained erythrocyte mass, and further work with this will eritrocitary in a cuvette with a built-in magnetic stirrer analyzer aggregation “Biola”, it's the same with constant stirring at a speed of "2" (temperature 37°C) after the tone, enter 0.3 ml of 0.85% sodium chloride solution. A calibration curve recorded for 2 minutes
Next, write the graph. To do this, in other dry cuvette containing a magnetic stir bar is placed 0.3 ml of erythrocyte suspension with constant stirring at a speed of "2" (temperature 37°C) it after the tone, enter 0.05 ml of 1% solution of acetic acid. Schedule record within 5 minutes
The first curve (1) reflects the dynamics of change in the average size of the particles (red blood cells) when exposed to acid hemolytica. Curve at first, gradually growing up, reflecting, thus, the process of stretching of red blood cells, which under the influence of hydrogen protons penetrating into the cells, there is an increase in oncotic pressure within them and, as a consequence, the increase of the entering current of water molecules. Having reached the peak, the curve begins to descend, which suggests that the process of fragmentation and complete destruction of erythrocytes under the influence of acid hemolytica.
The second curve (2) characterizes the change in the value of transmittance of the laser beam when passing through the erythrocyte in the, the specific point in time, the growth stops and the curve reaches a plateau.
The reading of the results.
To evaluate the acid resistance of erythrocytes using the following parameters.
For curve 1:
1) the initial average size of erythrocytes (srvc.ed.);
2) the maximum average size of erythrocytes (srvc.ed.);
3) the elongation of erythrocytes - magnitude increase in the average size of particles from the initial to the maximum (expressed in percent, with an initial average particle size is taken as 100%);
4) check the maximum average size of red blood cells (sec).
For curve 2:
1) the maximum rate of hemolysis (srvc.ed.);
2) check the maximum speed of hemolysis (s);
3) the average rate of hemolysis is a value expressed by the area bounded by:
the top curve 2;
on the right is the perpendicular from 2 curve at the point x-axis corresponding to time the third minute incubation of erythrocytes (srvc.ed.).
In conclusion, calculate the index of acid resistance (X), representing the parameters of both curves, reflecting the stability of the studied red blood cells to the effects of acid hemolytica. It is determined by the formula:
- gV m - the average rate of hemolysis (srvc.ed.), which is determined by the second curve.
In the study of blood samples of healthy people received the following normal values X: average of 0.59, with the range of normal fluctuations from 0.50 to 0.68.
The claimed method has the following advantages:
the simplicity and ease of execution
- the cost in time,
- parallel recording changes in the average size of red blood cells and the amount of light transmission.
The inventive method will be widely used in the clinic for the diagnosis of various anemias.
Evaluation method for acid resistance of erythrocytes by determining the amount of light transmission of standardized suspension of erythrocytes after adding acid hemolytica with graphing hemolysis, characterized in that the suspension of erythrocytes will standardize the breeding of erythrocyte mass saline solution in a ratio of 1:3 and centrifugation for 15 min, followed by dilution of 0.01 ml of erythrocyte sediment 4.0 ml of physiological solution, as the acid of hemolytica use 1% solution of acetic acid, conduct parallel change record with the maximum average size of erythrocytes and the average rate of hemolysis and calculate the index of acid resistance of erythrocytes (X) by the formula
X= t_d max / gVm,
where t_d max - time maximum mean radius of the cells;
gVm - average rate of hemolysis, the river. units;
the value of X from 0.50 to 0.68 consider normal.
SUBSTANCE: invention relates to laboratory methods for blood analysis. Plasma is dropped in copper sulfate solution with density 1.023 g/cm3, not above, and time for drop falling on bottom of graduated cylinder with column height 243 mm is measured. The blood plasma density value is calculated by the formula:
wherein is the unknown blood plasma density (g/cm3); is copper sulfate solution density measured by areometer (g/cm3); t is average falling time of plasma drop in the copper sulfate solution (as seconds); 0.260130126 and 0.00290695 are correction coefficients. Temperature of plasma and copper sulfate solution is 20oC. Method is simple and suitable and allows carrying out analysis of small volumes of blood plasma and to reduce analysis time.
EFFECT: improved assay method.
SUBSTANCE: method involves carrying out microscopic examination of blood serum samples taken from femoral vein and cubital vein. Femoral vein sample is taken on injured side. The examination is carried out before and after treatment. The blood serum samples are placed on fat-free glass slide in the amount of 0.01-0.02 ml as drops, dried at 18-30°C for 18-24 h. The set of pathological symptoms becoming larger or not changed after the treatment in comparison to sample taken before treatment, and morphological picture of samples under comparison taken from the cubital vein showing no changes or being changed to worse, the treatment is considered to be effective.
EFFECT: enabled medicamentous treatment evaluation in course of treatment to allow the treatment mode to be changed in due time; avoided surgical intervention (amputation); retained active life-style of aged patients.
FIELD: medicine, obstetrics, gynecology.
SUBSTANCE: in the first trimester of pregnancy one should study the content of CD8+CD11b lymphocytes and at their values being either equal or above 2% it is possible to predict gestosis. The present innovation enables to choose correct tactics of treating pregnant women that, in its turn, leads to decreased frequency of this complication of pregnancy and the risk for the development of fetal and neonatal pathology.
EFFECT: higher accuracy of prediction.
3 ex, 1 tbl
SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.
EFFECT: higher accuracy of detection.
FIELD: medicine, diagnostics.
SUBSTANCE: the present innovation deals with blood sampling, separating plasma against erythrocytes, moreover, in plasma on should detect activity of antithrombin III, proteins C and S, XIIa-dependent fibrinolysis and concentration of plasminogen obtained results should be expressed as relative units followed by calculating integral parameter that characterizes the state of anticoagulant-fibrinolytic potential (IPAFP) by the following formula: IPAFP = [(C1 + C2)/(C3 + C4)] x 100, where C1 - the ratio of observed value of antithrombin III activity to the value of inferior border of the range of analogous parameter norm; C2 - the ratio of observed value for the activity of proteins C and S system to the value of inferior border of the range of this parameter norm; C3 - the ratio of the value of inferior border of plasminogen concentration under normal conditions to observed value of analyzed parameter; C4 - coefficient calculated with the help of regression equation: C4 = 0.9 + (0.01 x X), where X - terms of lysis of patient's euglobulin clot/min, and at IPAFP value of 101.4 U and higher one should state anticoagulant-fibrinolytic blood potential to be in norm, in interval of 64.8 - 101.3 -as insufficient, and at 64.7 and below - as critical. The present method simplifies the procedure of evaluating the state of endogenous anticoagulants and activity of XIIa-dependent fibrinolysis.
EFFECT: increased diagnostic value of obtained results.
3 ex, 1 tbl
FIELD: medicine, laboratory diagnosis.
SUBSTANCE: method involves determination of the patient blood content of globulin-alpha 1, globulin-beta, globulin-gamma and the total bilirubin content followed by calculation of diagnosis indices for the patient (Y1, Y2, Y3) by using the computer program "Statistica 1.5" and introducing values X1, X2, X3 and X4 in computer wherein X1 means globulin-alpha 1 value; X2 means globulin-beta value; X3 means globulin-gamma value; X4 means total bilirubin value. Obtained values of diagnosis indices for the individual patient (Y1, Y2, Y3) are compared with average values of diagnosis indices (Y1', Y2', Y3') for different urogenital infections followed by comparison by sign and value. By the maximal coincidence of diagnosis index values for the individual patient with average diagnosis index values urogenital disease is diagnosed and the following diagnosis index average values are used: for chlamydiosis: Y1' = -2; Y2' = -0.1; Y3' = -0.2; for mycoplasmosis: Y1' = 2; Y2' = 0.8; Y3' = -0.04; for ureaplasmosis: Y1' = 2; Y2' = -1; Y3' = 0.02; for health persons: Y1' = -2; Y2' = 0.1; Y3' = 0.2. Invention provides the development of a method for express-diagnosis of infection at initial stage and diagnosis of atypical forms that occur in these diseases, and differential diagnosis of chlamydiosis, mycoplasmosis and ureaplasmosis. Invention can be used for carrying out the differential diagnosis of chlamydiosis, mycoplasmosis and ureaplasmosis.
EFFECT: improved method for express-diagnosis.
2 tbl, 4 ex
SUBSTANCE: method involves determining absolute value of ratio between lymphocyte number and absolute value of monocyte number in peripheral blood at the end of combine radiation therapy. The ratio is divided by 4.05. The result value being greater than 1, no disease relapse occurrence is predicted during the first observation year. The value being less than 1, tumor growth progress is stated and carcinoma relapse is predicted at the first year after treatment.
EFFECT: enhanced accuracy in detecting pathological process progress before observing clinical manifestations.
SUBSTANCE: method involves determining infrared radiation absorption coefficient in blood plasma in bandwidth of 1543-1396 cm-1. The infrared radiation absorption coefficient is determined in %. The value being equal to 29.7±1.1%, catarrhal cholecystitis is diagnosed. The value being 26.4±1.4%, phlegmonous cholecystitis is diagnosed. The value being 21.2±1.8%, gangrenous cholecystitis is diagnosed. The value being equal to 18.6±0.5%, gangrenous perforated cholecystitis case is diagnosed. The value in norm is equal to 32.4±0.8%.
EFFECT: high accuracy and specificity of diagnosis.
SUBSTANCE: the present innovation deals with biomedical measuring technologies, in particular, to those to detect bactericide activity of blood serum according to the level of its inhibiting impact upon luminescence intensity of sulfur-sensitive luminescent bacteria (ΣimpO) against control - luminescence intensity the same sulfur-sensitive luminescent bacteria that had no contact with blood serum (ΣimpK), then one should calculate the value of bactericide activity of blood serum by the following formula:
As sulfur-sensitive luminescent bacteria one should apply either natural or recombinant microorganisms being characterized by direct proportionality between intensity of decreased spontaneous bioluminescence level and degree of bactericide effect. For example, it is possible to apply Escherichia coli strain with genes of Photobacterium leiognathi luminescent system. The suggested method enables to shorten the duration for detecting bactericide activity of blood serum and decrease its labor intensity.
EFFECT: higher efficiency of detection.
1 cl, 1 ex, 1 tbl
SUBSTANCE: method involves determining blood insulin I and thyroxin T content and phagocytic leukocyte activity (PLA). Activity coefficient is calculated on the basis of formula KA=IxPLA/T. KA value being found greater than 2.8 units, considerable amelioration treatment effect is predicted. The value being from 1.4 to 2.8 units, amelioration is predicted. KA being less than 1.4 units, lower treatment efficiency is predicted.
EFFECT: high reliability of prognosis.