RU2726207C1 - Method for combined measurement of concentration of peroxidase-positive cells (neutrophilic granulocytes) and sperm cells in human ejaculate using variations based on cytochemical staining - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, particularly to a method for investigating ejaculate for diagnosing spermatogenesis and infertility disorders. Method for examining an ejaculate for diagnosing spermatogenesis and infertility disorders involves collecting ejaculate, preparing the preparation for examination, preparing a Goryaev counting chamber, depositing cells, determining the number of large squares of the chamber required for counting leukocytes and sperm cells, wherein for fixation and dilution of ejaculate used is a single working solution, which is prepared by mixing developer and 10 mcl of 30 % hydrogen peroxide, as developer used is a mixture of disodium phosphate dibasic mixture; monosubstituted potassium phosphate; ammonium chloride; disodium salt of ethylenediaminetetraacetic acid and benzidine, preparation for analysis is prepared by placing into a test tube of ejaculate and working solution at dilution of at least 1:10, mixed and incubated at temperature 37 °C for 30 minutes, wherein colouring leukocytes in brown, and sperm cells, spermatogenesis cells, lymphocytes, monocytes, macrophages, spermiophages, histiocytes, erythrocytes, lipoid bodies, the epithelium remains colourless, the preparation is filled with two sides of the Goryaev counting chamber for double counting of coloured leukocytes and spermatozoa with subsequent determination of the average value, Gorayev counting chamber is held in horizontal position for at least 4 minutes at room temperature for cell sedimentation, concentration of sperm cells and leukocytes in squares of Goryaev counting chamber is determined, ejaculate is coloured with eosin solution, 5 % aqueous solution and nygrosine solution, 10 % aqueous to determine the number of dead red-coloured and live uncoloured spermatozoa, staining of ejaculate with solution of azure-eosin dye according to Romanovsky and fixator-dye eosin methylene blue according to May Grunwald for detecting sperm cells, using data obtained as a result of ejaculate analysis for diagnosis.EFFECT: method described above enables to obtain analysis results with high diagnostic accuracy, information value and reliability.9 cl, 6 dwg

Description

FIELD OF THE INVENTION

The invention relates to medicine and can be used in the analysis of biological materials, in particular for the diagnosis of disorders of spermatogenesis and infertility in urology, andrology, oncology during the functional test of spermatozoa to assess their fertilizing ability and to identify the etiopathogenesis of pathozoospermia.

State of the art

Assessment and calculation of the concentration of neutrophilic granulocytes / peroxidase-positive leukocytes in the ejaculate is carried out using cytochemical staining with ortho-toluidine (see the WHO Guidelines for the Study and Processing of Human Ejaculate. - CAPITAL PRINT Publishing House, Moscow, 2012, fifth edition, ISBN 978-5 -905106-09-05 (section 2.18.1.5-1.7; Fig. 2.14; 3.11).

The disadvantages of this method are as follows:

1. there is no commercially available kit for staining, reagent: as a rule, 6 reagents are offered, which are difficult to buy in view of the fact that they are precursors,

2. It is difficult to prepare for use, due to the fact that modern laboratories use, as a rule, exclusively ready-made sets of reagents and do not have the necessary attorney equipment: scales, titration equipment, etc.,

3. used ortho-toluidine is a particularly dangerous carcinogen, which causes a health risk to personnel,

4. the results are counted in an improved Neubauer hemocytometer, which is not available on the Russian laboratory market of equipment and supplies with the necessary registration certificate,

5. The calculation formula is adapted to the Neubauer hemocytometer, which causes certain difficulties.

A known method for assessing and calculating the concentration of neutrophilic granulocytes (leukocytes) / peroxidase-positive cells (PPC) in the ejaculate. In its implementation, a semi-quantitative assessment of the leukocyte content in the ejaculate on the test strip is used using the immunochromatographic method based on the enzymatic activity of leukocyte esterase (without cytochemical staining) (see V.V.Dolgov, S.A. Lugovskaya, N.D. and other Laboratory diagnosis of male infertility. - M. - Tver: LLC Triad Publishing House, 2006. - 145 p., 199 ill., 5-94789-144-1. Page 70).

The disadvantages of this method are as follows:

1. The method allows only an approximate determination of the concentration and only neutrophilic granulocytes / peroxidase-positive leukocytes in the ejaculate,

2. Does not give an accurate idea of the presence or absence of an inflammatory process in a man.

3. The method of determination is non-quantitative (semi-quantitative “on the test strip”:> or <1 × 10 ⁶ , which contradicts WHO recommendations.

4. Leukocyte esterase appears in the bioliquid during the destruction of leukocytes, which can lead to false positive diagnosis (pyospermia).

A known method for the quantitative assessment of the content of round cells (neutrophilic and other leukocytes, macrophages, spermatogenesis cells) (concentration) without cytochemical staining of peroxidase-positive cells / neutrophilic granulocytes (leukocytes) in the ejaculate (see EE Bratin. Protocol for conducting spermiological studies. - Andrology and genital surgery. - 2014, No. 1, p. 20).

The disadvantages of this method are as follows.

1. The concentration of only round cells (all in a row) is determined, and not only neutrophilic granulocytes / peroxidase-positive leukocytes in the ejaculate.

2. The method does not give an accurate idea of the presence or absence of an inflammatory process in a man.

3. The method is not acceptable for monitoring the content of leukocytes in semen.

4. The identification of leukocytes occurs by subjective assessment ("by eye") by indirect identification signs, which does not comply with WHO recommendations.

A known method for determining the concentration of sperm in 1 milliliter (ml) of ejaculate (see WHO Guidelines for the study and treatment of human ejaculate, CAPITAL PRINT publishing house, Moscow, 2012, fifth edition, ISBN 978-5-905106-09-05 (section 2.7 , 2.8.1, 2.8.4, p. 34-47).

The disadvantages of this method are as follows.

1. The calculation of the concentration is carried out only sperm in 1 milliliter (ml) of ejaculate.

2. Calculation is carried out in an improved Neubauer hemocytometer, which is not on the Russian laboratory market of equipment and supplies with a registration certificate.

3. The fixation and dilution of the sperm sample occurs with a working solution consisting of a complex prescription of chemicals - precursors (paragraph 2.7.5 of the WHO guidelines), which are not used in a modern clinical diagnostic laboratory. One of these precursors is a particularly dangerous carcinogen - formalin.

4. Evaluation of the required dilution for further calculation of sperm concentration is based on complex mathematical calculations and is also adapted to the Neubauer hemocytometer (WHO Guide, pp. 40-41 chapter 2.8.1).

5. All formulas for counting are adapted to the Neubauer hemocytometer.

Closest to the claimed method is a method for measuring the concentration of spermatozoa in 1 milliliter (ml) of ejaculate, adopted as a prototype (see EE Bratina. Protocol for conducting spermiological studies. - Andrology and genital surgery. - 2014, No. 1, p. 18-19).

According to the known method, the preparation of the preparation is carried out, the ejaculate is fixed, the counting chamber is prepared, the ejaculate is filled in the hemocytometer windows, the sperm is deposited in the chamber, the number of large squares required for sperm counting is measured, the sperm count in 1 milliliter (ml) of ejaculate in the chamber and making an opinion.

The disadvantages of this method are as follows.

1. Only measurement of sperm concentration in 1 milliliter (ml) of ejaculate is carried out.

2. The fixation and dilution of the sperm sample is carried out with several working solutions (50 g of NaHCO ₃ , 10 ml of 36-40% formaldehyde solution (saturated solution)). One of them is a precursor and is a particularly dangerous carcinogen formaldehyde. The fixation with physiological saline is long (until the mobile cells completely stop), which is reflected in the laboratory turn-around time / TAT (turn around time) and introduces additional errors.

3. Evaluation of the quality of the required dilution for further calculation of sperm concentration is multi-stage and complicated (p. 18: when preparing the preparation (10 μl, coverslip 22 × 22 mm), the depth of the preparation is approximately 20 μm. A special sperm count is used in the field of view to determine the optimal With a microscope field of view diameter (measured with a micrometer lens) of 500 μm, the detection of 4 spermatozoa in the field of view approximately corresponds to 1 million / ml If the field of view diameter of 250 μm - 1 sperm in the field of view corresponds to 1 million / ml. Table 2 follows after multiplying the number of sperm detected by 4).

4. The step of determining the number of large squares required for sperm counting also complicates the research process, which is reflected in the TAT

Thus, the known methods are low informative and unreliable, and accordingly the task is to create a reliable, simple, reliable and informative analysis technique.

SUMMARY OF THE INVENTION

The invention is aimed at solving the problem of increasing diagnostic reliability.

The technical result achieved by using the presented method consists in increasing the diagnostic accuracy of the analysis results, information content and reliability. The process of preparation and analysis of biological material is simplified.

The technical result is achieved due to the fact that in the method for the study of ejaculate, including the preparation of the drug for research and working solution, fixation of the ejaculate, preparation of the counting chamber of Goryaev, sedimentation of cells, determining the number of large squares required for counting leukocytes and sperm counts, counting the number of leukocytes and spermatozoa in the ejaculate and making a conclusion, use a single working solution for fixation and dilution, which is prepared by mixing the developer and 10 μl of 30% hydrogen peroxide, the preparation for the study is prepared by placing at least 1:10 in the tube of ejaculate and working solution in the tube, mix within 10 sec. and incubated at a temperature of 37 degrees for 30 minutes, the ejaculate is stained with a solution containing a developer and 10 μl of 30% hydrogen peroxide, while spermatozoa, spermatogenesis cells, lymphocytes, monocytes, macrophages, spermiophages, histiocytes, erythrocytes, lipoid bodies, epithelia colorless, the ejaculate is stained with a solution of eosin, 5% aqueous, and a nigrosin solution, 10% aqueous, while the dead spermatozoa are colored red, and the living remain unpainted, the ejaculate is stained with a solution of the azure-eosin stain according to Romanovsky and fixative dye eosin methylene blue according to May-Grunwald, with the nuclei of the cells staining blue-violet, the cytoplasm of granulocytes, eosinophils, erythrocytes staining pink, sperm heads blue with lilac or violet acrosomal part, sperm tails with soft lilac , cytoplasmic drop and sperm neck stained in blue-violet the second color, fill the drug with two sides of the Goryaev’s counting chamber for double counting of stained leukocytes with subsequent determination of the average value, hold the Goryaev’s counting chamber in a horizontal position for at least 4 minutes at room temperature to precipitate cells, and determine the sperm concentration in large squares of the Goryaev’s counting chamber.

The technical result is enhanced due to the fact that the determination of sperm concentration in large squares of the counting chamber of Goryaev is carried out using dilution in accordance with a preliminary visual assessment.

With a small number of round cells, evaluated by a preliminary visual assessment, the number of leukocytes is determined in 100 large squares of the Goryaev chamber and the calculation is carried out according to the formula:

L = (a × 250 × 10 × 1000) / (100 × 1,000,000 *) [mln / ml]

where L is the number of leukocytes in 1 ml of ejaculate, and is the number of leukocytes in 100 large squares.

With a large number of round cells, estimated by a preliminary visual assessment, the number of leukocytes is determined in 40 large squares of the Goryaev chamber and the calculation is carried out according to the formula:

L = (a × 250 × 10 × 1000) / (40 × 1,000,000) [ppm]

where a is the number of leukocytes in 40 large squares, 250 is the volume of one large square, 10 is the degree of dilution of sperm, 1000-1000 μl in 1 ml of ejaculate, 40 is the number of large squares.

With a small number of sperm, estimated by a preliminary visual assessment, the determination of sperm concentration is carried out in that side of the Goryaev’s chamber, where the leukocytes were counted at a dilution of 1:10.

Determination of sperm concentration in 5 large squares of the Goryaev chamber, divided into 16 small ones, is carried out diagonally according to the formula:

C = a × 500,000 [ppm],

where C is the concentration of sperm in 1 ml of ejaculate, and is the number of sperm in 5 large squares diagonally.

With a large number of sperm cells, estimated by a preliminary visual assessment, increase the dilution of the drug to 1:20, 1:40 or 1:80, adding 200 μl of the working solution to the test drug.

The sperm concentration with a large number of sperm, estimated by a preliminary assessment, is determined in 5 large squares of the Goryaev’s chamber, divided into 16 small squares, diagonally according to the formula:

C = a × 1,000,000 [ppm],

where C is the concentration of sperm in 1 ml of ejaculate, and is the number of sperm in 5 large squares diagonally

With a large number of sperm in the ejaculate, as estimated by a preliminary assessment, dilution is increased to 1:40, 1:50, 1:80 or 1: 100, each time adding 200 μl of the working solution.

With an increase in dilution, the free squares of the Goryaev chamber are filled to calculate sperm concentration.

When diluting the ejaculate in a proportion of 1:40 or more, the sperm concentration is determined in 5 large squares of the Goryaev’s chamber, divided into 16 small squares diagonally, according to the formula:

C = (a × 250 × P × 1000) / (40 × 1,000,000) [million / ml],

where C is the concentration of sperm in 1 ml of ejaculate

a - sperm count in 5 large squares diagonally

P - semen dilution rate,

As the developer using a mixture of sodium phosphate disubstituted; potassium phosphate monosubstituted; ammonium chloride; disodium salt of ethylenediaminotetraacetic acid and benzidine.

The quality control of the study is carried out, while whole blood is used as a positive control when determining the staining of leukocytes of ejaculate samples, staining results are evaluated by microscopy.

Description of graphic materials

The invention and the possibility of achieving a technical result will be more clear from the following description with reference to the figures of graphic materials, which show:

in FIG. 1 - 100 large squares of the Goryaev’s chamber, divided into 4 small,

in FIG. 2 - 40 large squares of Goryaev’s chamber, divided into 4 small,

in FIG. 3 - five large squares of the camera Goryaev, divided into 16 small,

in FIG. 4 - five large squares of the camera Goryaev, divided into 16 small,

in FIG. 5 - the results of a clinical examination according to the method adopted as a prototype,

figure 6 - the results of a clinical examination of the proposed method.

The implementation of the method

The diagnostic action of the presented method is based on the cytochemical staining method used for analysis of human sperm according to the WHO Guidelines for the study and processing of human sperm. The composition of the kit for implementing the method contains all the necessary components to standardize the performance of a routine spirogram in a clinical diagnostic laboratory. The analysis procedure consists of five stages using the following staining methods, the reagents for which are part of the kit:

- staining of sperm and peroxidase-positive cells (PPC) / neutrophilic granulocytes in the ejaculate;

- staining of live / dead sperm,

- staining in order to differentiate sperm morphology,

- liquefaction of a viscous ejaculate.

In the presented method, in contrast to the known methods, the determination of the two most important spermogram parameters is performed in one test tube. Fixation and breeding of a sperm sample is carried out by the upper respiratory tract, which is harmless to aspiration, and a non-carcinogenic solution of 30% hydrogen peroxide. Hydrogen peroxide 30% allows you to use a working solution of reagents both for counting the concentration of peroxidase-positive cells (PPC) at a dilution of sperm 1:10, and to fix moving cells for the subsequent calculation of the concentration of sperm when diluting ejaculate from 1:10 and higher in large squares Goryaev’s cameras using appropriate formulas.

At the same time, objective assessment of the required dilution for further calculation of sperm concentration is objective: “If the sperm are in monolayers (a small amount after a preliminary visual assessment), then the sperm concentration must be calculated in the same side of the Goryaev’s chamber, where leukocytes were counted, that is, in previous dilution 1:10.

If the sperm cells are in multilayers (a large number after a preliminary visual assessment), then it is necessary to increase the dilution to 1:20 or more by adding another 200 μl of the working solution to test tube No. 1 with the test solution. If a dilution of 1:40, 1:80 is necessary, then it is necessary to add to the tube No. 1 with the existing dilution each time 200 μl of the working solution, respectively.

Variable formulas are presented for calculating the concentration of leukocytes and the concentration of spermatozoa, depending on the results of a preliminary, more objective assessment of the number of spermatozoa and dilution of ejaculate based on it.

The final diagnosis is made by the doctor after a complete clinical and laboratory examination of the patient.

When processing a sperm sample with a developer (reagent No. 1), 30% hydrogen peroxide (reagent No. 2), cells containing peroxidase (neutrophilic white blood cells) are stained brown. Peroxidase-free cells (spermatozoa, spermatogenesis cells, lymphocytes, monocytes, macrophages, spermiophages, histiocytes, erythrocytes, lipoid bodies, epithelium) do not stain (remain colorless).

Reagent No. 2 allows you to use a working solution of the reagent both for counting the concentration of peroxidase-positive cells (PPC) when sperm is diluted 1:10, and to fix mobile cells for the subsequent calculation of sperm concentration when diluting ejaculate from 1:10 and higher in large squares of the chamber Goryaev using the appropriate formulas.

Counting the concentration of round cells (CC) is excluded; CC includes cells of spermatogenesis of various maturity, neutrophilic leukocytes (granulocytes), lymphocytes, monocytes, macrophages, spermiophages, histiocytes. Only a preliminary qualitative assessment of the presence of QC in the drug is required.

As a result of the interaction of sperm with Reagent No. 3 (Eosin Solution, 5%) water and Reagent No. 4 (Nigrosine Solution, 10% water), the background of the preparation turns dark gray, on which dead spermatozoa stained with eosin are clearly visible in red unpainted (colorless) live sperm, which allows for separate counting of live and dead sperm and determine their percentage.

Reagent No. 5 (dye azur-eosin according to Romanovsky) and Reagent No. 6 (fixative-dye eosin methylene blue according to May-Grunwald) specifically interact with various cell structures. In this case, acidophilic structures are stained in different tones of red, basophilic structures in tones from purple to blue. Due to the fact that different cellular structures have different pH and bind to the dye with the opposite reaction. Cell nuclei are rich in nucleic acids, have an acid reaction and are stained with azure-eosin Romanovsky dye in blue-violet color. The cytoplasm of granulocytes, the granularity of eosinophils, and erythrocytes contain alkaline proteins, therefore, they are stained with the azure-eosin dye according to Romanovsky in pink. Sperm heads are stained blue with a lilac or violet acrosomal part. Sperm tails are painted in a pale lilac color. The cytoplasmic drop and sperm neck are stained blue-violet.

Reagent No. 7 (Phosphate-saline solution of bromelain) is a solution for liquefying ejaculate.

The method is used for a comprehensive assessment of spermogram indicators.

The reagents for the comprehensive analysis of sperm include:

- Reagent No. 1 (The developer is a colorless transparent liquid, contains sodium phosphate disubstituted 12-water chemically concentrated 26.6 mmol; Potassium phosphate monosubstituted chemically pure 66.1 mmol; ammonium chloride 4.62 mmol; disodium salt of ethylenediaminetetraacetic acid 14 mmol EDTA - disodium salt of DISSOLVINE NA2, Benzidine 2.5 mmol);

- Reagent No. 2 (Hydrogen Peroxide 30%)

- Reagent No. 3 (Eosin solution, 5% aqueous);

- Reagent No. 4 (Nigrosine solution, 10% aqueous);

- Reagent No. 5 (Dye azur-eosin according to Romanovsky);

- Reagent No. 6 (Fixative-dye eosin methylene blue according to May-Grunwald);

- Reagent No. 7 (Phosphate-saline solution of bromelain).

The sensitivity and specificity of cell staining), as the percentage of samples corresponding to the passport value is not less than 100%.

The normal value of the concentration of neutrophilic leukocytes (AUC) according to the WHO Guidelines for the study and treatment of human sperm, 5th edition (2010) is not more than 1.0 × 10 ⁶ per 1 ml of ejaculate.

Excessive white blood cell count in the ejaculate (pyospermia) may be due to infection and poor sperm quality.

White blood cells can reduce sperm motility and disrupt DNA integrity through oxidative effects. However, the observed level of leukocyte damage depends on factors that cannot be assumed, such as the cause, time and anatomical location of the leukocyte infiltration, as well as the origin of the leukocytes and their presence in the activated state (Tomlinson et al., 1993; Aitken & Baker, 1995; Rossi & Aitken, 1997).

The appearance of leukocytes in the ejaculate in a concentration higher than the threshold is the reason for a more in-depth examination of the man.

The minimum reference sperm concentration, according to the WHO Guidelines for the Study and Processing of Human Sperm, 5th edition (2010), is considered to be 15 × 10 ⁶ sperm per 1 ml of ejaculate.

Fertilization and the onset of pregnancy may depend on the concentration of sperm in the semen, it is determined by the volume of secrets of the seminal vesicles and the prostate (Eliasson, 1975) and is not a special measure of testicular function.

By the presented method, all cells present in the sample are stained (interference is not observed if the requirements of the instructions for use are observed). Interpretation and subsequent counting of stained cells is carried out by a KLD doctor who has studied according to an appropriate professional development program lasting at least 18 hours (“Practical spermogram course, 18 hours”; “Spermogram. Fundamentals of cytology of ejaculate sediment, 18 hours”, “Analysis of human sperm, 36 hours” ”) In the direction of further professional education in the specialty 31.08.05“ Clinical Laboratory Diagnostics ”in educational institutions authorized to conduct the indicated programs can be found on the website www.ssc-school.com.

When implementing the method, conical graduated, polypropylene, non-sterile 15 ml tubes are used; glass subject thicknesses. 2.0 ± 0.2 mm; glass coverslips 24 × 24 mm; laboratory disposable smear spatulas; a solution for discharging used consumables; single-channel automatic dispensers of variable volume 10-100 μl; tips for single-channel automatic dispensers of variable volume 2-200 μl; medical diagnostic gloves, disposable (GOST R 52239); microscope; protective glasses; Goryaev’s camera; Pasteur pipettes; glass rod; laboratory counter; graduated cylinders with a capacity of 25-1000 ml; stopwatch, filter paper.

The determination is made from biological samples of a person (seminal fluid). The biological material "ejaculate" for staining with a set is recommended to be stored for no more than 12 hours at a temperature of 25 to 37 ° C.

To prepare the working solution, it is necessary: 10 μl of Reagent No. 2 (hydrogen peroxide 30%) is added to the vial with Reagent No. 1 (Developer) and mixed. The working solution can be used within 24 hours after preparation, during storage 0-5 degrees C. If the working solution cannot be used completely within the specified time (within 24 hours after preparation, during storage from 0 to 5 ° C), it is permissible to freeze aliquots of the working solution for 1 month, followed by a single thaw before conducting a subsequent study to stain white blood cells and counting their concentration.

Reagents No. 3-7 are ready for use.

If liquefaction of the ejaculate has not occurred within 60 minutes, an equal amount of Reagent No. 7 Phosphate-saline solution of bromelain (dilute the sample of ejaculate twice) is added to the tube with the entire portion of the ejaculate with a Pasteur pipette, stirred, avoiding the formation of bubbles. Incubate the tube in an incubator at 37 ° C for 10 minutes.

The liquefaction procedure can affect the biochemical parameters of the ejaculate, its motility and sperm morphology, so the use of the procedure must be recorded.

Double dilution should be taken into account when calculating the concentration of sperm and white blood cells by multiplying the results by 2.

To stain the AUC in the ejaculate, the sperm sample is thoroughly mixed, avoiding the formation of bubbles and foam. An aliquot of 20 μl of sperm and 180 μl of a working solution of a working solution of reagents 1-2 (dilution 1:10) is placed in a test tube — a finished mixture of the test drug (Test tube No. 1).

Stir the suspension in a test tube for 10 seconds. glass rod and incubated at room temperature for 30 minutes

After preliminary re-mixing of the suspension, two sides of the Goryaev chamber are filled for double counting of leukocytes with subsequent determination of the average value (in order to minimize the error). To precipitate cells, the Goryaev chamber is held horizontally for at least 4 minutes at room temperature.

The filled chambers are evaluated in a light microscope at a magnification of × 200 or × 400.

Neutrophilic white blood cells, stained brown. contain peroxidase.

Peroxidase-free cells (spermatozoa, spermatogenesis cells, lymphocytes, monocytes, macrophages, spermiophages, histiocytes, erythrocytes, lipoid bodies, epithelium) do not stain brown (remain colorless).

Count the number of PPC in the squares of the Goryaev chamber using a laboratory counter.

The calculation of the concentration of leukocytes / PPC is carried out in two ways

The first option is if round cells (CC) are located in monolayers (not a large / small amount after a preliminary visual assessment), white blood cells (PPC) are counted in 100 large squares of the Goryaev’s chamber.

The calculation is carried out according to the formula:

L = (a × 250 × 10 × 1000) / (100 × 1,000,000 *) [mln / ml]

- объем одного большого квадрата, 10 - степень разведения спермы, 1000-1000 мкл в 1 мл эякулята, 100 - количество больших квадратов (фиг. 1).where L is the number of leukocytes (AUC) in 1 ml of ejaculate, and is the number of leukocytes (AUC) in 100 large squares, 250 -

- the volume of one large square, 10 - the degree of dilution of sperm, 1000-1000 μl in 1 ml of ejaculate, 100 - the number of large squares (Fig. 1).

A shortened version of the formula:

L = a / 40 [ppm]

* When converted to million / ml (WHO units), the result must be divided by 1,000,000.

In FIG. 1 shows 100 large squares of Goryaev’s chamber, divided into 4 small ones.

Example: when counting in 100 large squares of the Goryaev’s camera, 35 PPK were found. The calculation of the concentration of leukocytes (PPC) in 1 ml of semen L = 35/40 = 0.875 million / ml.

2nd option for calculating the concentration of PPC

If round cells (CC) are located in multilayers (a large number after a preliminary visual assessment), then cells can be counted in 40 large squares (20 squares at the top of the grid and 20 at the bottom). The calculation is carried out according to the formula:

L = (a × 250 × 10 × 1000) / (40 × 1,000,000) [ppm]

- объем одного большого квадрата, 10 - степень разведения спермы, 1000-1000 мкл в 1 мл эякулята, 40 - количество больших квадратов (фиг. 2).where a is the number of white blood cells (PPC) in 40 large squares, 250 -

- the volume of one large square, 10 - the degree of dilution of sperm, 1000-1000 μl in 1 ml of ejaculate, 40 - the number of large squares (Fig. 2).

A shortened version of the formula:

L = a × 0.0625 [ppm]

Example: when counting in 40 large squares of the Goryaev’s camera, 25 PPK were found. The calculation of the concentration of leukocytes (PPC) in 1 ml of semen L = 25 × 0.0625 = 1.5625 million / ml.

In FIG. 2 shows 40 large squares of Goryaev’s chamber, divided into 4 small ones.

Calculation of the concentration of sperm in the ejaculate

1. If spermatozoa lie in monolayers (a small amount after a preliminary visual assessment), then the concentration of sperm is carried out in the same side of the Goryaev’s chamber, where leukocytes were counted, that is, in the previous dilution of 1:10.

2. Assess the filling of the camera in a light microscope at a magnification of × 200 or × 400.

3. Count the number of sperm using a laboratory counter.

4. At least two sides of the Goryaev camera are counted in order to achieve an acceptable low error.

The calculation is carried out according to the 1st option:

1st variant of sperm count

When diluting the ejaculate 1:10, the sperm concentration is determined in 5 large squares (Fig. 3) of the Goryaev’s chamber, divided into 16 small ones, diagonally. The calculation is carried out according to the formula:

C = a × 500,000 [ppm]

where C is the concentration of sperm in 1 ml of the shark, and is the number of sperm in 5 large squares diagonally

In FIG. 3 shows 5 large squares of Goryaev’s chamber, divided into 16 small ones.

Example: when counting in 5 large squares on the diagonal of the Goryaev’s chamber, 135 spermatozoa were detected. Calculation of the concentration of sperm in 1 ml of sperm C = 135 × 500,000 = 67.5 million / ml.

If the sperm cells are in multilayers (a large amount after a preliminary visual assessment), it is necessary to increase the dilution to 1:20 or more by adding another 200 μl of the working solution to test tube No. 1 with the test solution. If a dilution of 1:40, 1:80 is necessary, then it is necessary to add to the tube No. 1 with the existing dilution each time 200 μl of the working solution, respectively.

With additional dilution of the working solution to calculate the concentration of sperm, it is necessary to fill in the new free chambers of Goryaev.

After preliminary careful re-mixing, the suspensions fill the Goryaev chamber. To precipitate cells, the Goryaev chamber is held horizontally for at least 4 minutes at room temperature.

The filled chamber is evaluated under a light microscope at a magnification of × 200 or × 400.

Count the number of sperm using a laboratory counter.

At least two sides of the Goryaev camera are counted in order to achieve an acceptable low error.

The calculation is carried out according to the 2nd option for calculating the concentration of sperm:

When diluting the ejaculate 1:20, the sperm concentration is also counted in 5 large squares (Fig. 4) of Goryaev’s chamber, divided into 16 small ones, diagonally. The calculation is carried out according to the formula:

C = a × 1,000,000 [ppm]

where C is the concentration of sperm in 1 ml of ejaculate, and is the number of sperm in 5 large squares diagonally

In FIG. 4 shows 5 large squares of Goryaev’s chamber, divided into 4 small ones.

Example: when counting in 5 large squares on the diagonal of the Goryaev’s chamber, 135 spermatozoa were detected. Calculation of the concentration of sperm in 1 ml of sperm C = 135 million / ml.

Next, a differential diagnosis of live and dead spermatozoa (Blum stain) of ejaculate samples is carried out:

For this, the ejaculate is thoroughly mixed. Apply 1 drop on a glass slide with a glass rod. 2 drops of an eosin solution are applied nearby (Reagent No. 3 Solution of eosin, 5% aqueous). A drop of ejaculate is mixed with a solution of eosin and incubated for 5 seconds. Add 3 drops of a nigrosin solution (Reagent No. 4 Nigrosine solution, 10% aqueous), mix and incubate for 5 seconds. A thin smear is prepared with a laboratory spatula.

The smear is dried and microscopic at a magnification of × 400 (lens × 40, eyepiece × 10).

As a result of painting:

- dead sperm when stained with reagents 3 and 4 are colored in red.

- live sperm cells when stained with reagents 3 and 4 do not stain (remain colorless).

Next, staining of sperm samples of ejaculate according to Romanovsky:

Make smears on slides, dry in air or in a thermostat at a temperature of no higher than 37 ° C until the wet shine disappears. Fix the dried preparations with an undiluted solution of fixer-dye according to May-Grunwald (Reagent No. 6 Fixer-dye eosin methylene blue according to May-Grunwald) - pour undiluted solution of fixer-dye so that it covers the entire smear for 1-2 minutes. Flushing of the retainer before painting is not required.

A dye working solution is prepared immediately before the smears are painted: a dye solution is mixed (Reagent No. 5 Azure-eosin dye according to Romanovsky) with de-ionized / distilled water in a ratio of 1 + 4 (1: 5) (per 10 ml of dye 40 ml of water). The resulting working solution of the dye can be stored up to 5-7 days in a closed container. A dye working solution is poured onto fixed smears of the ejaculate, after 15-20 minutes the preparations are washed with de-ionized / distilled water, dried and microscopic.

If the fixation time with the fixative-dye is exceeded, it can excessively dry on a blood smear and precipitate dot crystals of the dye, which are visible in the field of view of the microscope and interfere with microscopic examination. When this effect appears, it is necessary to reduce the fixation time.

Staining results are evaluated by microscopy.

Sperm heads when stained with reagents 5 and 6 are colored blue with a lilac or violet acrosomal part.

Sperm tails when stained with reagents 5 and 6 are painted in a pale lilac color.

The cytoplasmic drop and neck of the sperm when stained with reagents 5 and 6 are colored in blue-violet.

Accounting for results. Quality control.

Whole blood is used as a positive control when determining the color of the shaped elements of ejaculate samples. Whole blood should be used for no more than 24 hours after collection (when stored for more than 2 hours, stored at a temperature of 2 to 8 ° C; storage temperature of 20 to 25 ° C is allowed for less than two hours; freezing is not allowed) and should not contain no additives like anticoagulants. The test blood will contain various types of white blood cells, including brown ones in this reaction. Some will be unpainted. Thus, the same preparation of the control sample will have both positive controls — brown-colored leukocytes (granulocytes), and negative controls — unpainted leukocytes. For a separate negative control, an analysis is performed without the use of peroxide. In this protocol, the blood sample will contain unpainted white blood cells.

As an example, in FIG. 5 and FIG. 6 shows the actual results of practical clinical research by the method adopted as a prototype and by the proposed method. The purpose of the examination is to determine the cause of infertility.

Comparison of the survey results obtained in accordance with the known and presented methods (Fig. 5 and 6), allows us to make the following conclusion:

1) An analysis of sperm (spermograms) of a 39-year-old male from 10/01/2019, performed at the clinic, revealed insignificant changes in sperm motility - “Asthenozoospermia”.

2) Analysis of sperm (spermogram) of the same man 39 years old from 10/05/2019, performed by experts of the International School of Cytology in the presented way revealed:

deeper fertility disorders

men: "Oligostenoteratozoospermia. Necrosoospermia"

the presence of inflammation, which can be the cause of male infertility, as well as evidence of the asymptomatic course of the human carcinogenic virus papilloma virus (hrHPV): "Pyospermia"

The study of the presented method allowed to appoint a qualified treatment.

Thus, the application of the presented method allows to eliminate the disadvantages inherent in the known methods, the presented method has the following advantages:

1. Provides the determination of the two most important spermogram parameters in one test tube, which increases the diagnostic performance and objectivity of the control.

2. The fixation and dilution of the sperm sample are carried out by the upper respiratory tract, which is harmless to aspiration, and not a carcinogenic solution of 30% hydrogen peroxide.

3. The reagent No. 2 used in the proposed method (hydrogen peroxide 30%) allows you to use a working solution of the reagent both for counting the concentration of peroxidase-positive cells (PPC) at a dilution of sperm 1:10, and to fix moving cells in order to subsequently calculate the concentration spermatozoa with dilution of the ejaculate from 1:10 and higher in the large squares of the Goryaev’s chamber, using the appropriate formulas, which increases the objectivity and reliability of the diagnostic results.

4. Objective assessment of the required dilution for further calculation of sperm concentration:

- If the sperm cells are in monolayers (a small amount after a preliminary visual assessment), then the sperm concentration must be calculated in the same side of the Goryaev’s chamber, where the leukocytes were counted, that is, in the previous dilution of 1:10.

- If the sperm cells are in multilayers (a large number after a preliminary visual assessment), then it is necessary to increase the dilution to 1:20 or more by adding another 200 μl of the working solution to test tube No. 1 with the test solution. If a dilution of 1:40, 1:80 is necessary, then it is necessary to add to the tube No. 1 with the existing dilution each time 200 μl of the working solution, respectively.

5. Variable models are presented for calculating the concentration of leukocytes and the concentration of spermatozoa depending on the results of a preliminary, more objective assessment of the number of spermatozoa and dilution of ejaculate based on it, which increases the objectivity and reliability of the diagnostic results.

Claims (20)

1. A method for the study of ejaculate for the diagnosis of disorders of spermatogenesis and infertility, including collecting ejaculate, preparing the drug for research, preparing the Goryaev counting chamber, sedimenting cells, determining the number of large squares of the chamber necessary for counting leukocytes and spermatozoa, - for fixation and dilution of the ejaculate using a single working solution, which is prepared by mixing the developer and 10 μl of 30% hydrogen peroxide, as a developer using a mixture of sodium phosphate disubstituted; potassium phosphate monosubstituted; ammonium chloride; disodium salt of ethylenediaminotetraacetic acid and benzidine, - the preparation for the study is prepared by placing in the tube the ejaculate and the working solution at a dilution of at least 1:10, mix for 10 seconds. and incubated at a temperature of 37 ° C for 30 minutes, while the white blood cells are stained brown, and spermatozoa, spermatogenesis cells, lymphocytes, monocytes, macrophages, spermiophages, histiocytes, erythrocytes, lipoid bodies, epithelia remain colorless, - fill the drug with two sides of the Goryaev’s counting chamber for double counting of stained white blood cells and sperm cells with subsequent determination of the average value, keep the Goryaev’s counting chamber in a horizontal position for at least 4 minutes at room temperature to precipitate cells, - determine the concentration of sperm and white blood cells in the squares of the counting chamber of Goryaev, - perform staining of the ejaculate with a solution of eosin, 5% aqueous and a solution of nigrosin, 10% aqueous to determine the number of dead, stained red, and living remaining unpainted sperm, - perform ejaculate staining with a solution of the dye azur-eosin according to Romanovsky and fixative-dye eosin methylene blue according to May-Grunwald to identify sperm structures, use the data obtained as a result of the study of ejaculate for diagnosis. 2. The method according to p. 1, characterized in that the determination of the concentration of sperm and leukocytes in large squares of the counting chamber of Goryaev is carried out using dilution in accordance with a preliminary visual assessment. 3. The method according to p. 2, characterized in that with a small number of sperm, estimated by a preliminary visual assessment, the determination of sperm concentration is carried out on the side of the Goryaev’s chamber where leukocytes were counted at a dilution of 1:10. 4. The method according to p. 2, characterized in that the determination of sperm concentration in 5 large squares of the Goryaev chamber, divided into 16 small squares, is carried out diagonally during dilution of the ejaculate: working solution 1:10 according to the formula C = a × 500,000 [ppm], where C is the concentration of sperm in 1 ml of ejaculate, and is the number of sperm in 5 large squares diagonally. 5. The method according to p. 2, characterized in that with a large number of sperm cells, evaluated by a preliminary visual assessment, increase the dilution of the drug to 1:20, 1:40 or 1:80, adding 200 μl of the working solution to the test drug. 6. The method according to p. 2, characterized in that the concentration of sperm with a large number of sperm, estimated by preliminary assessment, is determined in 5 large squares of the Goryaev’s chamber, divided into 16 small squares, diagonally when diluted ejaculate: working solution 1:20 according to the formula C = a × 1,000,000 [ppm], where C is the concentration of sperm in 1 ml of ejaculate, and is the number of sperm in 5 large squares diagonally. 7. The method according to p. 2, characterized in that with a large number of sperm in the ejaculate, as estimated by a preliminary assessment, increase the dilution to 1:40, 1:50, 1:80 or 1: 100, each time adding 200 μl of working solution . 8. The method according to p. 5 or 7, characterized in that when the dilution is increased to calculate the concentration of sperm, the free squares of the Goryaev chamber are filled. 9. The method according to one of paragraphs. 1-8, characterized in that they monitor the quality of the study, while whole blood is used as a positive control when determining the color of leukocytes of ejaculate samples, the staining results are evaluated by microscopy.

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