Taking a biomaterial smear. Collection, storage and delivery of material for PCR studies

Instructions for collection, storage and transportation

biological material for laboratory research

for the detection of pathogens by PCR

1.1.1. The biomaterial is taken from the expected habitat of microorganisms and the development of infection.

1.1.2. The amount of material taken should be small. Excess discharge, mucus and pus adversely affect the quality of DNA extraction and contribute to DNA degradation during storage and transportation.

1.1.3. When introducing a biomaterial taken from a patient with a probe with a cotton swab or brush into an Eppendorf tube with a buffer solution, it is necessary:

Observe sterility;

Before immersing the biomaterial collected on the tampon (brush) into the solution, smear it on the dry wall of the test tube, then moisten the tampon (brush) in the solution and, rotating the probe, thoroughly wash off all the material from the wall of the test tube and the tampon (brush);

If possible, squeeze the swab against the wall of the test tube, remove the probe with the swab (brush) and close the test tube.

1.2. Methods for taking biomaterial for research by PCR

1.2.1. Scrapings. For scrapings, disposable sterile probes are used that have a cotton swab with increased adsorption or “brushes”, less often Volkmann spoons, small ear spoons or similar instruments with slightly blunt edges. Scraping movements to collect material. The test material should contain as many epithelial cells as possible and a minimum amount of mucus, blood and exudate impurities. Transfer the material to a sterile Eppendorf tube with buffer solution (see section 1.1.).

Scraping from the cervical canal: before taking a scraping, it is necessary to remove excess mucus with a sterile cotton swab, treat the cervix with sterile saline. Insert the probe into the cervical canal to a depth of 0.5-1.5 cm, avoiding contact with the walls of the vagina, and collect the material with a scraping movement (not to blood). A small number of red blood cells in the sample does not affect the result of the analysis. In the presence of cervical erosion, the material is taken from the border of healthy and altered tissue.

Scraping from the urethra: insert the probe into the urethra (in men - to a depth of 2-4 cm) and collect the material with several rotational movements. On the eve before taking the material, a provocation is allowed (spicy food, alcohol, etc.). It is recommended to refrain from urinating for 1-2 hours before sampling. In the presence of abundant purulent discharge scraping should be taken no later than 15 minutes after urination.

Scraping from the conjunctiva take, after having previously anesthetized the eye with a 0.5% solution of dicaine. Turning the eyelid, use a probe with a cotton swab (or eye scalpel) to collect epithelial cells from the conjunctiva.

Scraping from the rectum: insert the probe into the anus to a depth of 3-4 cm and collect the material with a rotational movement.

1.2.2. Strokes. Using a disposable sterile probe with a cotton swab or brush, take a small amount of discharge (from the vaults of the vagina, from the pharynx, nasopharynx, etc.) and transfer it to a sterile Eppendorf tube with a buffer solution (see section 1.1.).

1.2.3. Blood. For PCR testing, blood must be native(not curled up).

Collect blood aseptically, by venipuncture - into a branded test tube (2-3 ml) with EDTA anticoagulant ( heparin should not be used as an anticoagulant!), gently mix by gently inverting the tube.

Blood with EDTA is a biomaterial for the following research groups:

- genetic research(the moment of blood sampling does not matter).

- detection of infectious agents(the most informative are samples taken during chills, elevated temperature, i.e. presumably during viremia or bacteremia).

1.2.4. Urine. In an empty sterile container with a tight screw cap, collect the morning first or middle portion of urine in an amount of at least 10 ml. If urine sampling is carried out during the day, then before that the patient should not urinate for 1.5-3 hours.

1.2.5. Sputum. Sanitize in the morning oral cavity and throat (rinse with a solution of baking soda). Collect sputum in an empty sterile wide-mouthed vial.

1.2.6. Biopsy. The material should be taken from the zone of the alleged location of the infectious agent, from the damaged tissue or the area bordering on the damage. Place the biopsy in a sterile Eppendorf tube with buffer solution.

1.2.7. Saliva, gastric juice, cerebrospinal fluid, synovial fluid. Saliva (1-5 ml), gastric juice (1-5 ml), cerebrospinal fluid (1-1.5 ml) - place in an empty sterile test tube (container).

1.2.8. Prostate juice. After prostate massage, the juice is collected in an amount of 0.5-1 ml in an empty sterile test tube (container). If it is impossible to get juice, immediately after the massage, the first portion of urine is collected in an amount of not more than 10 ml (this portion contains prostate juice).

1.2.9. Washouts, bronchoalveolar lavage (BAL). With a sterile cotton swab and 5-7 ml of saline, a wash is carried out, for example, from the tip of an endoscope or bronchoscope, and 0.5-5 ml of wash is placed in an empty sterile tube (container).

1.2.10. Feces. A probe with a cotton swab is placed inside stool and turn slightly, capturing a small amount of material. Then the material is placed in a sterile Eppendorf tube with a buffer solution.

1.3. Rules for storing biological material for PCR research

1.3.1. Swabs, scrapings:

In a refrigerator (+4ºС - +8ºС) up to 7 days;

In the freezer (-20ºС) up to 14 days (only one defrosting is allowed!).

1.3.2. Urine: in the refrigerator (+4ºС - +8ºС) no more than 1 day;

1.3.3. Blood with EDTA: in a refrigerator (+4ºС - +8ºС) - to determine infectious agents - no more than 1 day; for genetic studies - up to 4 days. Freezing is not subject.

1.3.4. Sputum

1.3.5. Biopsies:

In the refrigerator (+4ºС - +8ºС) no more than 1 day,

In the freezer (-20ºС) up to 2 weeks.

1.3.6. prostate juice: in the refrigerator (+4ºС - +8ºС) no more than 1 day.

1.3.7. synovial fluid: in the refrigerator (+4ºС - +8ºС) no more than 1 day.

2. Rules for taking, storing and transporting blood

for immunochemical research

2.1.1. On the eve of blood sampling, exclude physical exercise, stressful situations, physiotherapeutic procedures, reception medicines(except in cases drug treatment prescribed by a doctor), oral contraceptives, alcoholic beverages and fatty foods. Do not smoke immediately before the study.

2.1.2. When examining the function thyroid gland during the period of treatment with drugs containing thyroid hormones, the study is carried out 24 hours after the last dose of the drug; 2-3 days before taking blood, exclude the use of drugs containing iodine.

2.1.3. When examining PSA a week before the analysis, exclude any manipulations with the prostate gland.

2.1.4. Blood is taken in the morning, on an empty stomach (to avoid chileness, i.e., turbidity of the serum), under conditions of physiological rest. Before taking blood, the patient must be given a 15-minute rest.

2.1.5. Blood sampling is carried out in the treatment room of the medical facility, in the position of the patient "sitting" or "lying", from the cubital vein in compliance with the rules of asepsis and antisepsis. Blood is collected in a sterile tube, mono-syringe or blood collection system (Vacutainer®, Vacuette®).

2.1.6. To obtain serum, venous blood settles until complete clotting. After the formation of a fibrin clot, the latter is separated from the walls with a sterile glass rod, strictly individual for each sample, the tube is centrifuged at 3000 rpm for 15 minutes * .

* The mono-syringe and blood collection systems (Vacutainer®, Vacuette®) are subjected to centrifugation without prior manipulations.

2.1.7. Supernatant yellow color(serum) is carefully pipetted into an empty tube with a cap (5 ml).

2.2. Rules for the storage of blood and blood serum for immunochemical studies

2.2.1. Tomoat: in the refrigerator (+40C - +80C) for 1 day.

2.2.2.Serumblood:

In the refrigerator at a temperature of + 40C - + 80C for 4 days;

In the freezer at -200C for 2 weeks.

Only one defrosting is allowed!

ATTENTION! Serum that does not contain impurities of erythrocytes, bacterial growths, chylosis, hemolysis is subject to research. In the presence of any of these signs, the serum is destroyed and repeated blood sampling is prescribed.

2.3. Rules for taking and storing blood for the study of lupus anticoagulant

and coagulation studies

2.3.1. Blood is taken in the morning on an empty stomach from the cubital vein in compliance with the rules of asepsis and antisepsis on the day of the study. To prevent activation of blood clotting, compression of the vein should not exceed 1 minute .

2.3.2. If the study is prescribed against the background of taking drugs that affect blood coagulation, it should be noted in the direction.

2.3.3. The blood is collected in a special tube containing sodium citrate, up to the mark. If this rule is not observed, the blood/anticoagulant ratio changes, which may adversely affect the accuracy of the result.

2.3.4. Within 1 hour after taking blood, the tube must be centrifuged at 3000 rpm for 15 minutes.

2.3.5. Plasma storage: up to 6 hours at +40C - +80C or up to 2 weeks at -200C.

2.4. Rules for taking and storing blood for the study of glycated (glycosylated) hemoglobin

2.4.1. Blood is taken in the morning on an empty stomach from the cubital vein, observing the rules of asepsis and antisepsis.

2.4.2. The blood is collected in a special tube containing EDTA, gently mixed. Do not centrifuge!

2.4.3. Delivery to the Laboratory on the day of blood sampling.

2.4.4. Storage: up to 2 weeks at -200C.

2.5. Prenatal Screening Rules

2.5.1. Optimal timing conducting the study: I ​​trimester - 10-13 weeks of pregnancy; II trimester - 16-18 weeks of pregnancy.

2.5.2. When referring to the study, a special direction is filled out, in which it is necessary to indicate the individual data of the pregnant woman: age (date / month / year), weight, ultrasound results (KTR, BDP, number of fetuses, gestational age according to ultrasound (weeks + days); if available - data on the size of the neck fold - NT) with the obligatory indication of the date of the ultrasound and the name of the doctor who performed the ultrasound; additional risk factors (smoking, diabetes, IVF), ethnicity.

2.5.3. For screening of the second trimester of pregnancy, you can use the data of an ultrasound scan performed in the first trimester.

3. Rules for taking, storing and transporting biomaterial

for bacteriological research

3.1. The biomaterial is taken before the course of antibiotic therapy is applied or not less than a week after its completion.

3.2. To prevent contamination of the sample by microorganisms from the external environment, the taken biomaterial is transferred to an empty container or test tube with a transport medium in compliance with the standard rules of sterility: the stopper of the test tube (container) is opened / closed so that the inside of the stopper remains sterile, the sterile glassware is not kept open for long.

3.3. Taking biomaterial for detection mycoplasmas and ureaplasmas, Trichomonas, gonococci, nonspecific microflora, crops anaerobic bacteria produced in branded test tubes with carbon transport medium:

Open the package in the indicated place according to the diagram;

Remove and open the test tube, discard the cap;

Use a probe to take a scraping / swab, biopsy or dip the probe into a biological fluid;

Place the probe with the biomaterial immediately into the test tube (the handle of the probe is the cap of the test tube);

Number the test tube, indicate the number in the direction.

3.4. Types of biological material for taking into the transport coal environment: smears / scrapings, prostate juice, cerebrospinal fluid, synovial fluid, biopsy.

3.5. Storage of biomaterial in test tubes with transport medium for detection mycoplasmas and ureaplasmas, nonspecific microflora until the moment of transportation -

Delivery to the Laboratory within one day.

3.6. Storage of biomaterial in test tubes with transport medium for detection Trichomonas, gonococci, cultures of anaerobic bacteria until the moment of transportation - at room temperature.

3.7. Native biomaterial: sputum, prostate juice, synovial fluid from 1 to 10 ml to detect non-specific microflora, mycoplasmas and ureaplasmas are collected in an empty sterile container.

It is necessary to notify the Laboratory on the eve or on the day of taking the biomaterial before 11:00.

Storage of native biomaterial until transportation - in the refrigerator compartment (+4ºС - +8ºС).

Delivery to the Laboratory on the day of sampling.

3.8. Urine to test for the degree of bacteriuria(morning medium portion, 10-20 ml) is collected in an empty sterile container with a lid after a thorough toilet of the external genitalia.

It is necessary to notify the Laboratory on the eve or on the day of taking the biomaterial before 11:00.

Storage of biomaterial until transportation - in the refrigerator compartment (+4ºС - +8ºС).

Delivery to the Laboratory on the day of taking the biomaterial.

3.9. Blood for sterility.

The most informative is the blood sample taken during the rise in temperature.

Blood is collected in branded bottles with a two-phase transport medium (bottle for adults, bottle for children). Carefully open the plastic cap of the bottle, wipe the part of the rubber stopper that has appeared with 70% alcohol. Under sterile conditions, take blood from a vein with a syringe and inject through a rubber stopper into a vial (4 ml - into a vial for adults, 2 ml - into a vial for children).

It is necessary to notify the Laboratory on the eve or on the day of taking the biomaterial before 11:00.

Storage of blood until transport - at room temperature.

Delivery to the Laboratory on the day of taking the biomaterial.

3.10. Breast milk to identify nonspecific microflora, it is taken from each breast into a separate sterile hermetically sealed cup. Sign the cups: “left breast”, “right breast”.

Before collecting the biomaterial, wash the chest with warm water and soap, wipe it with a clean towel, carefully treat the nipples and the peripapillary area with a cotton swab moistened with 70% ethyl alcohol solution, dry with a sterile napkin (each gland is treated with a separate swab). The first 10-15 ml of expressed milk are not used for analysis. Express the second portion of milk from each breast into the signed cup in the amount of ~ 10 ml.

It is necessary to notify the Laboratory on the eve or on the day of taking the biomaterial before 11:00.

Delivery to the Laboratory within 2 hours on the day of taking the material.

3.11. Feces for dysbacteriosis.

Before taking the sample, it is forbidden to drink alcohol for 3 days and take antibiotics for 2 weeks. The chair must be obtained without enemas and laxatives. Children with constipation can use a bar of soap for an irritating effect. Faeces are collected in an individual vessel, washed from the disinfectant. With a sterile spoon, 3-5 g of an average portion of feces are taken and placed in a sterile container.

It is necessary to notify the Laboratory on the eve or on the day of taking the biomaterial before 11:00.

Storage until the moment of transportation - store the container with the biomaterial to keep warm wrapped in cotton wool and paper.

Delivery to the Laboratory on the day of taking the biomaterial.

4. Rules for taking, storing and transporting biomaterial

for microscopic examinations

4.1. From each patient 2 glasses(one glass is spare). On every glass 3 points: vagina ( V), cervical canal ( FROM), urethra ( U). Apply strokes so that the matte surface is on the left, on top, and from it - from left to right: v, c, u.

4.2. The marking (No.) on the glass should not dissolve in alcohol (you can sign on the frosted part of the glass with a simple pencil). The smears should be fixed by air drying (30-60 min).

4.3. The glasses must be marked, folded with smears to each other, packed in an individual bag with the appropriate accompanying inscription (health center, full name of the patient, no. on the glass).

4.4. The package and completed referral are sent to the Laboratory.

5. Rules for taking, storing and transporting biomaterial

5.1. Within 24 hours before the study, it is necessary to exclude douching, the use of intravaginal therapy and sexual intercourse. You can not take the material during menstruation. The most informative are samples taken 14-20 days after the onset of menstruation.

5.2. The material is taken before a bimanual study, various diagnostic tests, before taking the material for PCR. The material is taken without pre-treatment vaginal mucosa.

5.3. A scraping from the vaginal/cervical mucosa is taken with a nylon Cervex-Brush, spatula, or other instrument and spread evenly over the slide, leaving one end of the slide clear (for marking). One point is taken on the glass, duplicated for one more spare glass (only two glasses per point). The smears should be fixed by air drying (30-60 min).

5.4. The glasses must be marked (No. and full name of the patient), folded with smears to each other, packed in an individual bag with the appropriate accompanying inscription (HCI, full name of the patient, No. on the glass).

5.5. Dried smears can be stored for up to 6 days at room temperature.

5.6. The package and the completed referral (one referral for each point indicating the type of biomaterial) are transferred to the Laboratory.

Do not put the direction in the glass bag, do not wrap the glass in the direction!

Department of Clinical Laboratory Diagnostics with the course of FPC and PC

The list of sections on the discipline of clinical laboratory diagnostics to prepare for the implementation of classroom tests in the winter laboratory-examination session by students of the 6th year of distance learning.

1. Organizational structure of the clinical diagnostic laboratory.

2. General clinical laboratory studies.

3. Hematological laboratory studies.

4. Laboratory methods assessment of hemostasis.

5. Biochemical laboratory research.

6. Quality control of laboratory research.

The list of control questions on the discipline of clinical laboratory diagnostics to prepare for the implementation of classroom tests in the winter laboratory-examination session by students of the 6th year of distance learning.

Structural organization of the clinical diagnostic laboratory.

Regulatory documents and orders of the Ministry of Health of the Republic of Belarus, regulating the work of the clinical diagnostic laboratory.

Clinical diagnostic laboratory equipment.

The main measuring and analytical equipment of the clinical diagnostic laboratory.

Fundamentals of safety precautions and rules of sanitary and epidemiological regime when working in a clinical diagnostic laboratory.

Rules for taking, delivering, receiving, processing and registering biological material.

General clinical laboratory research.

Preparation of the patient for the study, collection, storage and processing of material for general analysis urine.

Physical properties of urine: color, transparency, smell, pH, relative density of urine.

Chemical properties of urine, methods for determining total protein (qualitative and quantitative methods, methods of "dry chemistry").

Determination of glucose in urine using indicator paper and glucose oxidase method.

Rules for the preparation of a native preparation of urinary sediment.

Quantitative determination of elements of organized urine sediment.

Determination of elements of unorganized sediment.

Types of unorganized precipitation.

General clinical analysis of blood.

Characteristics of methods definitions of ESR, hemoglobin concentration, counting the number of red blood cells, color index, white blood cells and leukocyte formula.

Rules for preparing the patient, sampling, storage and processing of material for general clinical analysis blood.

Normal values ​​and limits of physiological fluctuations in the level of erythrocytes and leukocytes in peripheral blood.

Rules for preparing the preparation and counting in the Goryaev chamber.

Method for determination of blood hemoglobin by hemoglobincyanide method.

Modern concept of bone marrow hematopoiesis.

Percentage of leukocytes in peripheral blood. Diagnostic value of the leukocyte formula.

Laboratory studies characterizing the blood coagulation system.

The concept of the blood coagulation system.

primary hemostasis. Laboratory methods for the study of primary hemostasis.

Phases of coagulation hemostasis.

fibrinolysis.

anticoagulant system.

Laboratory methods for assessing secondary hemostasis.

Laboratory studies characterizing biochemical parameters blood.

Laboratory indicators characterizing protein metabolism. Determination of total protein in blood serum by the biuret method.

Residual nitrogen and its components. Methods for determining urea, creatinine, uric acid.

Methods for the study of enzymes. Clinical and diagnostic value of determining the activity of enzymes.

The biological role of carbohydrates. Determination of glucose content by enzymatic method.

Plasma lipoproteins. Laboratory indicators of lipid metabolism.

Formation and metabolism of bile pigments. Clinical and diagnostic significance of the study of pigment metabolism indicators.

Analytical reliability and quality control of clinical laboratory studies.

Analytical assessment of laboratory results. Types of errors, their characteristics.

Intralaboratory quality control. Methods for monitoring the reproducibility of research results. Control of the correctness of the results of the study.

Cleaving an analytical laboratory conclusion.

The analytical laboratory conclusion is drawn up according to the conditions of the situational problem.

Based on their analysis of the proposed laboratory data, the following questions should be answered:

The principle underlying the analyzed method;

Necessary equipment;

Rules for the preparation of the test material and the main sources of errors possible when performing this method;

Units;

The concept of referential values. Clinical and diagnostic value of the analyzed method.

Practical skills:

Organization of the workplace for laboratory research;

Preparation of reagents in the required concentration;

Reception, labeling and storage of biological material;

Performing a general blood test;

Performing a general urinalysis;

Performing a hemostasiological study;

Performing biochemical studies;

Registration of results of laboratory researches;

The procedure for taking clinical material into a test tube with a transport medium:

1. Open the vial cap.

2. Using a disposable sterile probe, obtain the discharge of the corresponding

biotope (vagina, urethra, cervical canal).

3. Transfer the probe with clinical material to a tube with a transport medium of 1.5 ml, when the probe hits the bottom of the tube, bend the thin part of the probe to the notch with additional force, then break it off and leave the probe in the tube. If necessary, obtain clinical material from several biotopes repeat the procedure, each time taking the clinical material with a new probe into a new tube.

3. Close the tube tightly with a lid, mark it.

Features of taking clinical material of the cervical canal:

1. Clinical material of the cervical canal is obtained after inserting a gynecological speculum into the vagina using a urethral probe or cervical cytobrush (a sufficient number of epithelial cells is needed for HPV testing!).

2. Before taking clinical material, it is necessary to carefully treat the opening of the cervical canal with a sterile gauze swab and then treat the cervix with sterile saline.

3. After inserting the urethral probe into the cervical canal by 1.5 cm, it is rotated several times (2-3 full turns clockwise) and removed. When removing the probe, it is necessary to completely exclude its contact with the walls of the vagina.

4. The obtained clinical material is placed in a test tube with a transport medium (see above).

Features of taking clinical material from the vagina:

On the day of the examination, women should not toilet the genitals and douche the vagina.

1. Clinical material from the vagina is obtained from the posterior or lateral fornix using a vaginal or urethral probe by scraping from the surface of the epithelium.

2. Clinical material must be obtained BEFORE the manual examination.

3. Before manipulation, the gynecological speculum can be moistened with warm water; the use of antiseptics for treating the speculum is contraindicated.

4. In girls (virgo), clinical material is obtained from the mucous membrane of the vestibule of the vagina without the use of a gynecological speculum.

5. The obtained clinical material is placed in a test tube with a transport medium (see above).

Features of taking clinical material from the urethra in women:

1. Clinical material from the urethra is obtained using a urethral probe.

2. Before taking the clinical material, the patient is advised to refrain from urinating for 1.5-2 hours.

3. If there is free urethral discharge, the external opening of the urethra should be cleaned with a cotton swab.

4. In the absence of free secretions, a light massage of the urethra can be performed.

5. After inserting the instrument into the urethra to a depth of 1 cm, it is necessary to advance it to the external opening, slightly pressing on the back and side walls of the urethra (rotational movements are painful).

6. The obtained clinical material is placed in a test tube with a transport medium (see above).

To study the material obtained from several biotopes, the procedure is repeated, each time using a new sterile probe and a new test tube!!!

It is unacceptable to mix material from the cervical canal, vaginal contents and urethra in one tube!

Features of taking clinical material from the urethra in men:

1.5–2 hours. To exclude distortions in the results of determining the composition of the microflora of the urogenital tract of men due to the presence of transient microflora in the urogenital tract, sexual abstinence or the use of protected sexual contact is recommended for three days before taking the biomaterial.

1. Before taking a scraping from the urethra, the head of the penis is treated in the area of ​​the external opening of the urethra with a swab moistened with sterile saline.

2.Massage the urethra. In the presence of secretions flowing freely from the urethra, they are removed with a dry swab.

3. The probe is inserted into the urethra to a depth of 1-2 cm. With several rotational movements, scraping of epithelial cells is performed and the probe is transferred into a test tube with a transport medium, broken off and left. Detachable is taken in sufficient quantity. A moderate presence of impurities in the form of mucus, blood and pus is acceptable.

Taking clinical material Withforeskin of the glans penis (HRP):

Before taking the biomaterial, the patient is advised to refrain from urinating for

1.5–2 hours.

1. Using a disposable probe, a scraping of epithelial cells is made from the corresponding biotope ( foreskin glans penis, preputial sac) and transfer the probe into a test tube with a transport medium, break off and leave.

Conditions for storage and transportation of biomaterial:

1. Tubes with received clinical material must be labeled.

2. In the accompanying document-direction, it is necessary to indicate: full name, age of the patient (s), clinical material, proposed diagnosis, indications for examination, date and time of sampling, name of the institution (department) sending the clinical material.

3. If the time of transportation of clinical material from the moment of taking to the moment of its delivery to the laboratory is not more than a day, then the tube with clinical material must be stored and delivered to the laboratory at the temperature of a domestic refrigerator (+ 4 + 10 ° C), without freezing.

Rules for taking, storing and transporting biomaterial

For the detection of pathogens by PCR

1.1.1. The biomaterial is taken from the expected habitat of microorganisms and the development of infection.

1.1.2. The amount of material taken should be small. Excess discharge, mucus and pus adversely affect the quality of DNA extraction and contribute to DNA degradation during storage and transportation.

1.1.3. When introducing a biomaterial taken from a patient with a probe with a cotton swab or brush into an Eppendorf tube with a buffer solution, it is necessary:

Observe sterility;

Before immersing the biomaterial collected on the tampon (brush) into the solution, smear it on the dry wall of the test tube, then moisten the tampon (brush) in the solution and, rotating the probe, thoroughly wash off all the material from the wall of the test tube and the tampon (brush);

If possible, squeeze the swab against the wall of the test tube, remove the probe with the swab (brush) and close the test tube.

Methods for taking biomaterial for research by PCR

1.2.1. Scrapings. For scrapings, disposable sterile probes are used that have a cotton swab with increased adsorption or “brushes”, less often Volkmann spoons, small ear spoons or similar instruments with slightly blunt edges. Scraping movements to collect material. The test material should contain as many epithelial cells as possible and a minimum amount of mucus, blood and exudate impurities. Transfer the material to a sterile Eppendorf tube with buffer solution (see section 1.1.).

Scraping from the cervical canal: before taking a scraping, it is necessary to remove excess mucus with a sterile cotton swab, treat the cervix with sterile saline. Insert the probe into the cervical canal to a depth of 0.5-1.5 cm, avoiding contact with the walls of the vagina, and collect the material with a scraping movement (not to blood). A small number of red blood cells in the sample does not affect the result of the analysis. In the presence of cervical erosion, the material is taken from the border of healthy and altered tissue.

Scraping from the urethra: insert the probe into the urethra (in men - to a depth of 2-4 cm) and collect the material with several rotational movements. On the eve before taking the material, a provocation is allowed (spicy food, alcohol, etc.). It is recommended to refrain from urinating for 1-2 hours before sampling. In the presence of abundant purulent discharge, scrapings should be taken no later than 15 minutes after urination.

Scraping from the conjunctiva take, after having previously anesthetized the eye with a 0.5% solution of dicaine. After turning the eyelid, use a probe with a cotton swab (or an eye scalpel) to collect epithelial cells from the conjunctiva.

Scraping from the rectum: insert the probe into the anus to a depth of 3-4 cm and collect the material with a rotational movement.

1.2.2. Strokes. Using a disposable sterile probe with a cotton swab or brush, take a small amount of discharge (from the vaults of the vagina, from the pharynx, nasopharynx, etc.) and transfer it to a sterile Eppendorf tube with a buffer solution (see section 1.1.).

1.2.3. Blood. For PCR testing, blood must be native(not curled up).

Collect blood aseptically, by venipuncture - into a branded test tube (2-3 ml) with EDTA anticoagulant ( heparin should not be used as an anticoagulant!), gently mix by gently inverting the tube.

Blood with EDTA is a biomaterial for the following research groups:

- genetic research(the moment of blood sampling does not matter).

- detection of infectious agents(The most informative are samples taken during chills, elevated temperature, i.e., presumably during viremia or bacteremia).

1.2.4. Urine. In an empty sterile container with a tight screw cap, collect the morning first or middle portion of urine in an amount of at least 10 ml. If urine sampling is carried out during the day, then before that the patient should not urinate for 1.5-3 hours.

1.2.5. Sputum. In the morning, sanitize the mouth and throat (rinse with a solution of baking soda). Collect sputum in an empty sterile wide-mouthed vial.

1.2.6. Biopsy. The material should be taken from the zone of the alleged location of the infectious agent, from the damaged tissue or the area bordering on the damage. Place the biopsy in a sterile Eppendorf tube with buffer solution.

1.2.7. Saliva, gastric juice, cerebrospinal fluid, synovial fluid. Saliva (1-5 ml), gastric juice (1-5 ml), cerebrospinal fluid (1-1.5 ml) - place in an empty sterile test tube (container).

1.2.8. Prostate juice. After prostate massage, the juice is collected in an amount of 0.5-1 ml in an empty sterile test tube (container). If it is impossible to get juice, immediately after the massage, the first portion of urine is collected in an amount of not more than 10 ml (this portion contains prostate juice).

1.2.9. Washouts, bronchoalveolar lavage (BAL). With a sterile cotton swab and 5-7 ml of saline, a wash is carried out, for example, from the tip of an endoscope or bronchoscope, and 0.5-5 ml of wash is placed in an empty sterile tube (container).

1.2.10. Feces. A probe with a cotton swab is placed inside the stool and slightly rotated, capturing a small amount of material. Then the material is placed in a sterile Eppendorf tube with a buffer solution.

If it is necessary to conduct a delayed analysis (transportation over long distances, technical malfunction of the device, etc.), blood samples are stored in a refrigerator (4 o - 8 o C) and examined within 24 hours. However, it should be taken into account that the cells swell and the parameters associated with their volume change. Practically healthy people these changes are not critical and do not affect the quantitative parameters, but in the presence of pathological cells, the latter can change or even collapse within a few hours from the moment of blood sampling.

Immediately before the study, the blood should be thoroughly mixed for several minutes to dilute the anticoagulant and evenly distribute the formed elements in the plasma. Long-term constant mixing of samples until the moment of their research is not recommended due to possible injury and decay of pathological cells.

A blood test on an automatic analyzer is carried out at room temperature. Blood stored in a refrigerator must first be warmed to room temperature, as blood viscosity increases at low temperatures and blood cells tend to stick together, which in turn leads to impaired mixing and incomplete lysis. The study of cold blood may cause the appearance of "alarm signals" due to compression of the leukocyte histogram.

When performing hematological studies at a considerable distance from the place of blood sampling, problems associated with unfavorable transportation conditions inevitably arise. The impact of mechanical factors (shaking, vibration, mixing, etc.), temperature conditions, the likelihood of spillage and contamination of samples can affect the quality of analyzes. To eliminate these reasons, when transporting blood tubes, it is recommended to use hermetically sealed plastic tubes and special isothermal transport containers. During transportation, contact of the sample (with native material) and the referral form is not allowed, in accordance with the rules of biological safety.

^ 4.3. Reception and registration of biological material

Tubes with venous blood samples are delivered to the laboratory on the day of taking them in racks in special bags for the delivery of biological material, in which the tubes must be in a vertical position, and when transported over a remote distance - in special containers.

The laboratory employee receiving the material must check:

The correctness of the referral: the referral form indicates the data of the subject (last name, first name and patronymic, age, case history or outpatient card number, department, diagnosis, therapy performed);

Labeling of test tubes with blood samples (they must be marked with the code or surname of the patient, identical to the code and surname in the form for sending the material for the study). The laboratory assistant must register the delivered material, note the number of test tubes.

^ 4.4 Counting cellular composition blood on a hematology analyzer

The calculation of the cellular composition of blood on a hematological analyzer must be carried out in strict accordance with the instructions for the device.

When working on a hematology analyzer, you should:

Use blood stabilized with K-2 EDTA in the recommended proportion for the specific salt and disposable hematology tubes (plain or vacuum);

Before analysis, carefully but thoroughly mix the tube with blood (it is recommended to use a device for mixing blood samples - rotomix for this purpose);

Use reagents registered in the prescribed manner; when changing reagents to products from another manufacturer, check and set the calibration of the instrument;

Start the device, observing all stages of flushing, achieving zero (background) values ​​of indicators for all channels;

Pay attention to instrument messages about possible systematic errors: remember that the increase in MCHC is higher normal values, usually the result of a measurement error;

If an error is detected, it is imperative to eliminate the cause, do not work on a faulty device;

Perform the quality control procedure according to the appropriate program. with an assessment of the result;

Work meaningfully, comparing the results obtained with clinical characteristic patient samples;

After the end of measurements, thoroughly rinse the analyzer;

When stopping the device for a long time, it is imperative to perform all conservation procedures (if possible, together with a service engineer);

In the event of technical problems, you should seek the help of a service engineer from a company licensed to Maintenance; Do not entrust work to untrained personnel.

When starting to work on a hematological analyzer, one should take into account the limits of linearity of measurement of the analyzed parameters. Evaluation of samples with values ​​of analyzed parameters that exceed the limit of measurement linearity can lead to erroneous results. In most cases, when analyzing samples with hypercytosis, the analyzer displays the “D” (dilute) icon instead of the value of the measured parameter, which indicates the need to dilute the sample and re-measurement. Dilution should be carried out until similar final results are obtained at the next two dilutions.

^ PRI me R ─ Analysis of a sample of a patient with hyperleukocytosis on three different hematological analyzers is presented in the table.

Table 3

¦Hyperleukocytosis counting results performed on three different hematology analyzers

1. 
   ^ Calibration of hematology analyzers

Which achieves equality to zero of the systematic component of the error (zero bias) or confirms that the bias is zero, taking into account the calibration error. Calibration of hematology analyzers can be performed in two main ways:

For whole blood;

Based on stabilized blood samples with certified values.

^ 5.1 Whole blood calibration

Whole blood calibration can be performed by comparison with a reference analyzer, the calibration of which has been verified and can be taken as the original.

For calibration by comparison with the reference analyzer, 20 patient venous blood samples are used, randomly selected from the daily research program of the laboratory that has the reference analyzer. Blood should be taken into vacuum tubes with anticoagulant K 2 EDTA. After analysis on the reference analyzer, the blood tubes are delivered to the laboratory for analysis on the device being calibrated. The time between measurements on the reference and calibrated analyzer should not exceed 4 hours. The analysis of calibration samples on the instrument to be calibrated must be performed in the same way as the analysis of patient samples. The results obtained are entered into a table in the form given in Appendix 1. These results are used to determine the appropriate calibration factors, which are numerically equal to the coefficients of the slope of the calibration straight lines passing through the origin on the graph.

Calibration factors are calculated on a computer using the EXCEL program included in the Microsoft OFFICE package of any version. To calculate the calibration factors in this program, a scatter plot is constructed, where measured values ​​are plotted along the X axis, reference values ​​are plotted along the Y axis, and a regression line is set through the origin. The program draws a calibration line and determines its slope, which is the desired calibration factor. If gross errors are detected - points that are significantly separated from the calibration line on the graph, they must be removed from the calculation (delete the entire line in the EXCEL table). The resulting calibration factor is entered in the table of Appendix 1 and is used to manually adjust the calibration of the device in accordance with its operating instructions.

1. 
   ^ Calibration against stabilized blood samples with certified values

Whenever possible, calibrators specifically designed by the manufacturer to calibrate a particular type of analyzer should be used. The errors of the set values ​​in modern hematological calibrators generally correspond to medical requirements. However, not all types of analyzers have such calibrators. In addition, due to the limited shelf life (usually no more than 45 days), it is not always possible to obtain samples that are not expired.

Control materials are not intended by the manufacturer for calibration purposes: tolerances for values ​​are much wider, validation and production control methods are not as strict as for calibrators. During calibration, control materials can be used only as one of the sources of information about the metrological properties of the device.

However, it should be borne in mind that when both control materials and calibrators are used for calibration purposes, the features of sample preparation and operation of the analyzers have a significant impact on the components of the systematic error. These influencing factors cannot be taken into account when calibrating with commercial materials. As a result, in all cases, the calibration performed must be verified by analyzing the statistics of the results obtained in patient samples (erythrocyte indices, intervals of normal values).

5.2.1 Calibration intervals for hematology analyzers

Calibration interval requirements are usually found in the analyzer's instruction manual. As a rule, calibration is required after a repair or when significant drift is detected in in-house quality control procedures.

5.2.2 Calibration procedure

The calibration procedure is performed in accordance with the analyzer's instruction manual.

5.2.3 Checking the calibration result

This control consists in the analysis of the calibration sample immediately after the calibration. The performed calibration is accepted if the results of the analysis correspond to the calibration values, taking into account the analytical variation of the analyzer.

5.2.4 Calibration verification using RBC indices

This method of checking and refining the calibration uses the fact that the average values ​​of erythrocyte indices - MCHC, MCH and MCV for patients have a rather small variation and therefore can be effectively used for calibration control and further quality control. The recommended number of samples for averaging is 20. Any patient sample can be used to determine the mean MCHC, while patients with anemia should be excluded from the mean MCHC and MCV.

Many modern hematology analyzers have built-in programs for calculating current averages, which greatly simplifies data processing.

The target values ​​of erythrocyte indices, averaged over 20 samples of patients aged 18 to 60 years, regardless of gender, are shown in Table 4.

Table 4

Target values ​​of erythrocyte indices

If the average values ​​obtained are outside the control tolerance, then this means that the MCV or Hgb or RBC calibration needs to be corrected.

When determining the reasons for an unsatisfactory calibration based on the results of the study of erythrocyte parameters when using commercial calibration / control materials, the following assessment of the reliability of the certified values ​​of the materials should be taken into account:

The most accurate and time-stable parameter is the concentration of erythrocytes;

Hgb is a stable parameter, but it may depend on the reagents used (for example, cyanide or cyanide-free);

The most unstable parameter is MCV. The MCV value tends to change during the entire shelf life of the material in a fairly wide range of values. When refining the MCV calibration, the data obtained from the analysis of the averages of the erythrocyte indices take precedence over the certified values ​​in the stabilized blood samples.

Assuming the RBC calibration is correct, Table 5 indicates possible reasons exit of the average values ​​of the indices beyond the control tolerance.

Table 5

Reasons for going beyond the average values ​​of erythrocyte indices