FS.3.2.0003.15 Human blood coagulation factor VIII. Coagulation factor VIII

The level of coagulation factor VIII from 0 to 1% causes an extremely severe form of the disease, from 1 to 2% - severe, from 2 to 5% - moderate, above 5% - light form, but with the danger of severe and even fatal bleeding in injuries and surgical interventions.

Among all possible manifestations hemophilia in the first place are hemorrhages in the large joints of the extremities (hip, knee, ankle, shoulder and elbow), deep subcutaneous, intermuscular and intramuscular hemorrhages, abundant and prolonged bleeding with injuries, the appearance of blood in the urine. Other bleeding is less common, including such severe and dangerous ones as retroperitoneal hemorrhages, hemorrhages in the abdominal organs, gastrointestinal bleeding, intracranial hemorrhages (strokes).

With hemophilia, one can quite clearly trace the progression of all manifestations of the disease as the child grows, and later on as an adult. At birth, more or less extensive hemorrhages under the periosteum of the skull bones, subcutaneous and intradermal hemorrhages, late bleeding from the umbilical cord. Sometimes the disease is detected at the first intramuscular injection, which can cause a large, life-threatening intramuscular hematoma. Teething is often accompanied by not very heavy bleeding. In the first years of life, there are often bleeding from the oral mucosa associated with trauma to various sharp objects. When a child learns to walk, falls and bruises are often accompanied by profuse nosebleeds and hematomas on the head. Hemorrhages in the orbit, as well as postorbital hematomas, can lead to loss of vision. In a child who has begun to crawl, hemorrhages in the buttocks are typical. Then, hemorrhages in the large joints of the limbs come to the fore. They appear the earlier, the more severe the hemophilia is. The first hemorrhages predispose to repeated outpourings of blood in the same joints. Everyone has it individual person, suffering from hemophilia, with particular persistence and frequency of hemorrhages, 1-3 joints are affected. The knee joints are most commonly affected, followed by the ankle, elbow, and hip joints. Hemorrhages in the small joints of the hands and feet (less than 1% of all lesions) and joints between the vertebrae are relatively rare. In each person, depending on the age and severity of the disease, from 1-2 to 8-12 joints are affected.

It is necessary to distinguish between acute hemarthrosis (primary and recurrent), chronic hemorrhagic-destructive osteoarthritis (arthropathy), secondary immune rheumatoid syndrome as a complication of the underlying process.

Acute hemarthrosis is manifested as sudden appearance(often after a minor injury) or a sharp increase in joint pain. The joint is often enlarged, the skin over it is red, hot to the touch. After the first transfusion of blood components, the pain quickly (within a few hours) decreases, and with the simultaneous removal of blood from the joint, it disappears almost immediately.

IV stages of joint damage are distinguished. In I, or early, stage, the volume of the joint may be increased as a result of hemorrhage. In the "cold" period, the function of the joint is not impaired, but X-ray examination determines the characteristic signs of the lesion. In stage II, the progression of the process is noted, which is revealed according to the data x-rays. In stage III, the joint sharply increases in size, deforms, often uneven and bumpy to the touch, pronounced hypotrophy of the muscles of the affected leg is determined. The mobility of the affected joints is more or less limited, which is associated both with damage to the joint itself and with changes in muscles and tendons. In this stage, pronounced osteoporosis is formed, fractures inside the joints easily occur. In the femur, there is a crater- or tunnel-like destruction of the bone substance typical of hemophilia. The patella is partially destroyed. Intra-articular cartilages are destroyed, mobile fragments of these cartilages are found in the joint cavity. Various kinds of subluxations and displacements of bones are possible. In stage IV, the function of the joint is almost completely lost. Joint fractures are possible. With age, the severity and prevalence of damage to the articular apparatus progresses and becomes more severe when hematomas occur around pathologically altered joints.

Secondary rheumatoid syndrome (Barkagan-Egorova syndrome) is a common form of joint damage in patients with hemophilia. For the first time, this syndrome was described in 1969. In many cases, it is viewed by doctors, since it occurs against the background of already existing hemarthrosis and destructive processes characteristic of hemophilia in the joints. Secondary rheumatoid syndrome is accompanied by a chronic inflammatory process (often symmetrical) in the small joints of the hands and feet, which were not previously affected by hemorrhages. Subsequently, as the process progresses, these joints undergo a typical deformation. AT large joints severe pain periodically appears, pronounced morning stiffness in the joints can be noted. Regardless of the appearance of new hemorrhages, the articular process is steadily progressing. At this point, a blood test reveals the appearance or sharp increase in existing laboratory signs. inflammatory process, including immunological ones.

In most people with hemophilia, the syndrome appears over the age of 10-14 years. By the age of 20, its frequency reaches 5.9%, and by 30 - up to 13% of all cases of the disease. With age, the prevalence and severity of all joint lesions are steadily progressing, which leads to disability, forcing the use of crutches, wheelchairs and other devices. The progression of joint damage depends on the frequency of acute hemorrhages, the timeliness and usefulness of their treatment (it is very important to conduct an early transfusion of blood and its components), the quality of orthopedic care, correct application physical therapy, physiotherapeutic and balneological effects, choice of profession and a number of other circumstances. All of these issues are currently extremely relevant, as life expectancy in hemophilia has increased dramatically due to the success of corrective treatment.

Extensive and intense subcutaneous, intermuscular, subfascial and retroperitoneal hematomas are very difficult and dangerous. Gradually increasing, they can reach enormous sizes, contain from 0.5 to 3 liters of blood or more, lead to the development of anemia, cause compression and destruction of the surrounding tissues and the vessels that feed them, necrosis. For example, retroperitoneal hematomas often completely destroy large areas pelvic bones(the diameter of the destruction zone can reach 15 cm or more), hematomas on the legs and arms destroy the tubular bones, the calcaneus. The death of bone tissue leads to the formation of hemorrhages under the periosteum. The process of such bone destruction on radiographs is often mistaken for a tumor process. Often, calcium salts are deposited in hematomas, which sometimes leads to the formation of new bones, which can close the joints and completely immobilize them.

Many hematomas, putting pressure on the nerve trunks or muscles, cause paralysis, sensory disturbances, and rapidly progressive muscle atrophy. Particularly dangerous are extensive hemorrhages in the soft tissues of the submandibular region, neck, pharynx and pharynx. These hemorrhages cause narrowing of the upper respiratory tract and suffocation.

serious problem with hemophilia, they create profuse and persistent renal bleeding, observed in 14-30% of people with this blood disease. These bleedings can occur both spontaneously and in connection with injuries of the lumbar region associated with pyelonephritis. In addition, renal bleeding may occur due to increased excretion of calcium in the urine due to the destruction of bone tissue in hemophilia. The appearance or intensification of such bleeding can be facilitated by taking analgesics (acetylsalicylic acid, etc.), massive blood and plasma transfusions, which leads to additional damage to the kidneys. Renal bleeding is often preceded by prolonged urinary excretion of blood particles that can only be detected by laboratory testing.

The appearance of blood in the urine is often accompanied by severe urination disorders, as well as a change in the amount of urine excreted (there may be both an increase in its daily volume and a decrease), attacks of renal colic due to the formation of blood clots in urinary tract. These phenomena are especially intense and pronounced during treatment, when the normal state of the blood is temporarily restored. The cessation of excretion of blood in the urine is often preceded by renal colic, and often a temporary absence of urine output with the appearance of signs of intoxication of the body with toxic metabolic products.

Renal bleeding periodically recurs, which over the years can lead to severe dystrophic-destructive changes in this organ, secondary infection and death from development kidney failure.

Gastrointestinal bleeding with hemophilia, they can be spontaneous, but more often they are caused by the intake of acetylsalicylic acid (aspirin), butadione and other drugs. The second source of bleeding are obvious or hidden ulcers of the stomach or duodenum, as well as erosive gastritis of various origins. However, sometimes there are diffuse capillary bleeding without any destructive changes in the mucous membrane. These bleedings are called diapedetic. When they appear, the intestinal wall is saturated with blood for a long time, which quickly leads to coma as a result of severe anemia, fainting state in connection with sharp decline blood pressure and death. The mechanism of development of such bleeding remains unclear to date.

Hemorrhages in the abdominal organs mimic various acute surgical diseases - acute appendicitis, intestinal obstruction and etc.

Hemorrhages in the head and spinal cord and their membranes in hemophilia are almost always associated either with injuries or with the use of drugs that disrupt the function of platelets, which are directly involved in blood clotting. Between the moment of injury and the development of hemorrhage there may be a light interval lasting from 1-2 hours to a day.

characteristic feature hemophilia are prolonged bleeding during injuries and operations. lacerations much more dangerous than linear breaks. Bleeding often does not occur immediately after injury, but after 1-5 hours.

Removal of the tonsils in hemophilia is much more dangerous than abdominal surgical interventions.

Extraction of teeth, especially molars, is often accompanied by many days of bleeding not only from the tooth sockets, but also from hematomas formed at the site of tissue infiltration with novocaine, which leads to the development of anemia. These hematomas cause destruction of the jaw. With hemophilia, teeth are removed against the background of the action of antihemophilic drugs under general anesthesia. Extraction of several teeth is best done at once.

Part of the complications in hemophilia is due to blood loss, compression and destruction of tissues by hematomas, infection of hematomas. A large group of complications is also associated with immune disorders. The most dangerous of them is the appearance in the blood in a large number of immune inhibitors (“blockers”) of blood coagulation factor VIII (or IX), transforming hemophilia into the so-called inhibitory form, in which the main method of treatment is transfusion therapy (transfusion of blood or its components) - almost completely loses its effectiveness. Moreover, repeated administration of antihemophilic drugs often causes a rapid increase in the amount of inhibitor in the blood, as a result of which the transfusion of blood and its components, which initially had some effect, soon becomes useless. The frequency of the inhibitory form of hemophilia, according to different authors, ranges from 1 to 20%, more often from 5 to 15%. With inhibitory forms, platelet function is noticeably impaired, hemorrhages in the joints and excretion of blood in the urine become more frequent, joint damage is significantly higher.

The main method of treatment and prevention of bleeding and hemorrhage of any localization and any origin in hemophilia is the intravenous administration of sufficient doses of blood products containing factor VIII. Factor VIII is variable and practically not preserved in canned blood, natural and dry plasma. For replacement treatment, only direct blood transfusions from a donor and blood products with preserved clotting factor VIII are suitable. Direct blood transfusions from a donor are resorted to only when the doctor does not have any other antihemophilic drugs. It is a grave mistake to transfuse blood from the mother, as she is a carrier of the disease, and the level of factor VIII in her blood is sharply reduced. In view of short period life of factor VIII in the blood of the recipient (about 6-8 hours), blood transfusions, as well as transfusions of antihemophilic plasma, should be repeated at least 3 times a day. Such blood and plasma transfusions are unsuitable for stopping massive bleeding and reliable cover for various surgical interventions.

An equal volume of antihemophilic plasma is approximately 3-4 times more effective than fresh banked blood. A daily dose of 30-50 ml / kg of body weight of antihemophilic plasma allows for some time to maintain a 10-15% level of factor VIII. The main danger such treatment is circulatory volume overload, which can lead to the development pulmonary edema. The use of antihemophilic plasma in a concentrated form does not change the situation, since a high concentration of the injected protein causes an intensive movement of fluid from the tissues into the blood, as a result of which the volume of circulating blood increases in the same way as when plasma is infused in a normal dilution. Concentrated dry antihemophilic plasma has only the advantage that it contains more concentrated factor VIII of blood coagulation, and in a small volume it is more quickly introduced into the bloodstream. Dry antihemophilic plasma is diluted with distilled water before use. Treatment with antihemophilic plasma is sufficient to stop most acute hemorrhages in the joints (except the most severe ones), as well as to prevent and treat minor bleeding.

The most reliable and effective in hemophilia concentrates of factor VIII of blood coagulation. The most accessible of them is cryoprecipitate. It is a protein concentrate isolated from plasma by cooling (cryoprecipitation), which contains a sufficient amount of blood clotting factors, but few proteins. Low content proteins allows you to enter the drug into the bloodstream in a very large quantities and increase the concentration of factor VIII to 100% or more without fear of circulatory overload and pulmonary edema. Cryoprecipitate must be stored at -20°C, which makes it difficult to transport. When thawed, the drug quickly loses its activity. Dry cryoprecipitate and modern concentrates of factor VIII of blood coagulation are deprived of these shortcomings. They can be stored in a regular refrigerator. Excessive administration of cryoprecipitate is undesirable, since it creates a high concentration of coagulation factors in the blood, as a result of which microcirculation in the organs is disturbed and there is a risk of blood clots and the development of DIC.

All antihemophilic drugs are administered intravenously only by stream, in the most concentrated form and as soon as possible after they are reopened without mixing with other solutions for intravenous administration. One of the main reasons for failure replacement therapy consists in the drip administration of blood products, which does not lead to an increase in the level of coagulation factor VIII in plasma. Until a stable stop of bleeding, you can not use any blood substitutes and blood products that do not contain antihemophilic factors, as this leads to a dilution of factor VIII and a decrease in its concentration in serum.

In case of acute hemorrhages in the joints, temporary (no more than 3-5 days) immobilization (immobilization) of the affected limb in a physiological position, heating of the affected joint (compresses), but not cooling is necessary. early removal the blood poured into the joint not only immediately eliminates the pain syndrome, prevents further blood clotting in the joint, but also reduces the risk of development and rapid progression of osteoarthritis. To prevent and treat secondary inflammatory changes after removal of blood, 40-60 mg of hydrocortisone is injected into the joint cavity. Supportive transfusion therapy, which is carried out during the first 3-6 days, prevents further bleeding and allows you to start physiotherapy exercises early, which contributes to a faster and more complete recovery of the function of the affected limb, and prevents muscle atrophy. It is better to develop movements in the affected joint in stages. In the first 5-7 days after removing the bandage, active movements are performed both in the affected joint and in other joints of the limb, gradually increasing the frequency and duration of exercises. From the 6-9th day, they switch to "load" exercises, using bicycle ergometers, pedal gates for hands, elastic traction. From the 11-13th day, in order to eliminate residual stiffness and limit maximum flexion or extension, passive load exercises are performed with caution. Simultaneously with the 5-7th day, physiotherapy is prescribed - hydrocortisone electrophoresis, anodic galvanization.

With hemorrhages in soft tissues, more intensive treatment with antihemophilic drugs is carried out than with hemorrhages in the joints. With the development of anemia, intravenous infusions of erythrocyte mass are additionally prescribed. If there are signs of infection of the hematoma, then broad-spectrum antibiotics are immediately prescribed. Any intramuscular injections with hemophilia are contraindicated, as they can cause extensive hematomas and pseudotumors. Penicillin and its semi-synthetic analogues are also undesirable, since in large doses they increase bleeding.

Early and intensive treatment with antihemophilic drugs contributes to the rapid regression of hematomas. Encapsulated hematomas are removed, if possible, surgically along with the capsule.

External bleeding from damaged skin, nosebleeds and bleeding from wounds in the oral cavity are stopped both by transfusion therapy and by local influences - by treating the bleeding area with drugs that promote blood clotting. In addition, these drugs can be taken orally. Pressure bandages or sutures are applied to the wounds. Similarly, stop bleeding after tooth extraction. When removed chewing teeth a slightly more intensive transfusion therapy is carried out, and the simultaneous removal of several teeth (3-5 or more) requires the introduction of antihemophilic drugs in the first 3 days.

In case of nosebleeds, tight packing should be avoided, since after the removal of the tampons, bleeding often resumes with even greater force. A quick stop of nasal bleeding is usually provided by antihemophilic plasma and antihemophilic drugs and simultaneous irrigation of the nasal mucosa with solutions that promote blood clotting.

A serious danger is represented by renal bleeding, in which intravenous infusions of antihemophilic plasma and cryoprecipitate are ineffective.

Gastrointestinal bleeding is controlled with large doses of clotting factor concentrates. It should be remembered that gastric bleeding is often provoked by taking aspirin, brufen, indomethacin in connection with pain in the joints, toothache or headache. In patients with hemophilia, even single dose aspirin can cause stomach bleeding.

In the prevention and treatment of chronic osteoarthritis and other lesions of the musculoskeletal system, it is necessary to provide various ways joint protection and prevention of limb injuries. To do this, foam rubber shields are sewn into the clothes around the knee, ankle and elbow joints, avoid those sports that involve jumping, falling and bruising (including cycling and motorcycle riding). The importance is given to the early and full treatment acute hemorrhages in the joints and muscles, intensive year-round physiotherapy exercises. For this, there are special complexes of atraumatic exercises in water, on soft mats and load devices - bicycle ergometers, manual gates. Classes should start in preschool or junior school age, i.e., before severe disorders of the musculoskeletal system developed. Complex therapy supplement with physiotherapeutic (currents high frequency, electrophoresis of glucocorticosteroids) and balneological methods of treatment, primarily mud therapy, brine and radon baths. With frequent and stubbornly recurring hemorrhages in the same joints, X-ray therapy is performed and surgery.

Minimizing the risk of injury and cuts from early childhood is important in the prevention of hemorrhage. Easily breaking toys (including metal and plastic ones), as well as unstable and heavy objects, are excluded from everyday life. Furniture should be with rounded edges, the protruding edges are wrapped with cotton wool or foam rubber, the floor is covered with a pile carpet. Communication and games of patients with girls are preferable, but not with boys. important for the patient right choice professions and places of work.

Prevention of hemophilia has not yet been developed. Determination of the sex of the unborn child by genetic research cells obtained from amniotic fluid, allows you to terminate the pregnancy in a timely manner, but does not show whether the fetus is a carrier of the hemophilia gene. Pregnancy is preserved if the fetus is male, since all the sons of the sick are born healthy. Terminate pregnancy if the fetus is female, since all daughters of hemophilia patients are carriers of the disease.

In female conductors of hemophilia who have a 50% chance of giving birth to an affected child (if the fetus is male), or who are transmitters of hemophilia (if the fetus is female), the birth of only girls transfers the risk of having hemophilia patients in the family from the first generation to the second, at the same time increasing the total number of carriers of the disease.

Low hemoglobin. Cholelithiasis. No seizures No competently prescribed treatment I'm asking for help. I have gallstone disease. Small pebbles and not much. Superficial gastritis. Non-closure of cardia and reflux of bile. Previously, there was also Helicobacter ++. Cured him. I’m afraid to remove the bile, maybe problems with casting will remain, everything will corrode the esophagus. Became lethargic lately. Shortness of breath and tachycardia. Blood pressure 100/60 and pulse 95. brittle hair and nails. Did a blood test. hemoglobin and serum iron and some other indicators of iron below the norm. I take omez. We have not had a consultation with a hematologist yet. The record is huge. I really want to understand what to do and how to be treated correctly. Help me please. There is no strength to be in such a state. Now I don’t know what examinations and treatment to undergo, one can harm the other. According to the ultrasound of the abdominal cavity, all the norms except for the bile. Indicators asat and alat seem to be normal, although they are increased. How can I get away from something.

Russian name

Latin name of substances Blood coagulation factor VIII + Willebrand factor

Pharmacological group of substances Blood coagulation factor VIII + Willebrand factor

Model clinical and pharmacological article 1

Characteristic. Factor Efficiency coagulation VIII determined on the basis of the International Standard for concentrate (FVIII:C), the effectiveness of the von Willebrand factor is determined on the basis of the determination of the effectiveness of the ristocetin cofactor (VW:RK), based on the International Standard for concentrate in accordance with the European Pharmacopoeia. The specific activity of the drug is not less than 60 IU FVIII:C/mg and not less than 53 IU FV:RK/mg of total protein. The number of units of blood coagulation factor VIII is expressed in IU, corresponding to the WHO standards for these drugs. Activity is expressed either as a percentage (relative to normal plasma levels of the factor) or in International Units (relative to the International Standard for Plasma Factor VIII). 1 IU of coagulation factor VIII is equivalent to the amount of coagulation factor VIII in 1 ml of normal human plasma.

Pharma action. Blood coagulation factor VSH binds to von Willebrand factor; activated factor VSH is a cofactor for activated factor IX, accelerating the conversion of clotting factor X into active form; activated factor X activates the conversion of prothrombin to thrombin, thrombin, in turn, activates the conversion of fibrinogen to fibrin, followed by the formation of a thrombus. The drug contains von Willebrand factor, which is a normal component of human plasma and acts in the same way as endogenous, and corrects hemostasis disorders in patients with von Willebrand disease: restores platelet adhesion to the vascular subendothelium at the site of vessel damage (attached to the vascular subendothelium and to the platelet membrane, provides primary hemostasis, reduces clotting time, while the effect is immediate and depends on the degree of protein polymerization); normalizes the concomitant deficiency of factor VIII (when administered intravenously, it binds endogenous factor VSH and stabilizes it, slowing down its rapid degradation), restores the level of factor VIII:C to normal. Replacement therapy with the drug in patients with hemophilia A increases the level of blood coagulation factor VIII, providing a temporary correction of factor deficiency and reducing the risk of bleeding. In addition, the von Willebrand factor promotes platelet adhesion in the area of ​​vascular damage and plays significant role during platelet aggregation.

Pharmacokinetics. von Willebrand disease type 3: the average recovery of VW:RK and VW:Ag - 68-99%, respectively, which corresponds to an average increase in plasma concentration of 1.5 and 2.1% per substituted IU / kg of body weight. T 1/2 FV: RK - 17.5 h, clearance - 3.9 ml / h / kg. Hemophilia A: the concentration of factor VIII: C is 80-120% of the expected value. T 1/2 VIII: C - 14.8 hours, which corresponds to the biological T 1/2, clearance - 2.9 ml / h / kg.

Indications. Treatment and prevention of bleeding in patients with von Willebrand disease (with quantitative and / or qualitative deficiency of von Willebrand factor), congenital hemophilia A or acquired deficiency of blood coagulation factor VIII.

Contraindications. Hypersensitivity, age up to 6 years (efficacy and safety not established).

Carefully. Pregnancy, lactation.

Dosing. In / in, after dissolution with the attached solvent; the resulting solution contains 90 IU of coagulation factor VIII and 80 IU of von Willebrand factor in 1 ml.

von Willebrand disease: the dose and duration of replacement therapy depends on clinical condition patient, type and severity of bleeding, level of FVIII:C and VW:RK.

The ratio between FVIII:C and FV:RK is 1:1, on average 1 IU/kg. FVIII:C and VW:RK increase the plasma level by 1.5-2% of the normal activity of the corresponding protein. Usual dose drug 20-50 IU/kg, which increases the level of FVIII:C and VW:RK up to 30-100%. The initial dose may be increased to 50-80 IU/kg, especially in patients with von Willebrand disease type 3, with gastrointestinal bleeding.

To prevent bleeding, it is necessary to start the administration of the drug 30 minutes before the start of the surgical intervention. In the case of a planned surgical intervention, the drug is administered 12-24 hours and 1 hour before the start of the surgical intervention, while the expected concentration of VW: RK is 60 IU / dl or more (60% or more) and FVIII: C is 50 IU / dl or more (50% or more). The dose is administered every 12-24 hours. Long-term treatment may cause an excessive increase in the level of FVIII:C. After 24-48 hours of treatment, in order to avoid an excessive increase in the level of FVIII:C, it is necessary to reduce the dose, or increase the interval between injections.

Hemophilia A: 1 IU of coagulation factor VIII:C/kg increases the factor in plasma by 1.5-2% of the normal content. Determination of the required dose: body weight (kg) × desired increase in the level of clotting factor VIII (%) × 0.5 IU / kg.

In the case of the bleeding events described below, the FVIII:C activity level should not fall below baseline plasma levels (% of normal) in the appropriate time period.

Moderate bleeding (early hemarthrosis, intramuscular bleeding, nosebleeds, oral bleeding and other minor injuries) - the required concentration of clotting factor VIII is 20-40 IU / dl (20-40%), the introduction is repeated every 12-24 hours, at least within 1 day, until the pain subsides or the source of bleeding heals.

More extensive bleeding (IM bleeding or hematoma) - the required concentration of coagulation factor VIII is 30-60 IU / dL (30-60%) every 12-24 hours for 3-4 days, until pain subsides and recovery is restored.

Life-threatening bleeding (intracranial, intraperitoneal, neck, blunt trauma, without a visible source of bleeding) - the required concentration of clotting factor VIII is 60-100 IU / dl (60-100%) every 8-24 hours, until the threat disappears completely.

Minor surgical interventions (including extraction of teeth) - the required concentration of clotting factor VIII is 30-60 IU / dL (30-60%) every 24 hours, for at least 1 day, until healing.

Major surgery - Required factor VIII levels (before and after surgery) - 80-100 IU/dl (80-100%) every 8-24 hours until wound heals adequately, then at least 7 days to maintain factor VIII activity at the level of 30-60%.

For long-term prevention of bleeding in patients with severe hemophilia A, it is necessary to administer 20-40 IU / kg every 2-3 days. In some cases, especially in young patients, it may be necessary to reduce the interval between injections or increase the dose.

If there is no effect from an adequate dose, or if it is impossible to achieve the desired plasma concentration of clotting factor VIII with adequate administration, it is necessary to conduct a Bethesda test for the presence of inhibitory antibodies to clotting factor VIII. In patients with high level inhibitors, treatment with factor VIII may be ineffective, and other therapeutic measures may be required.

Side effect. allergic reactions(urticaria, skin rash, chills, feeling of pressure in the chest, shortness of breath, decreased blood pressure, rarely - anaphylactic shock), burning at the injection site, hyperthermia, "hot flashes", headache, drowsiness, apathy, nausea, vomiting, anxiety. The development of inhibitory antibodies to von Willebrand factor - in von Willebrand's disease, in hemophilia A - to blood coagulation factor VIII (usually IgG), which leads to an inadequate clinical response to the administration of the drug; inhibitory antibodies can cause precipitation and contribute to the development of anaphylactic reactions.

In patients receiving von Willebrand factor preparations containing coagulation factor VIII, a prolonged increase in plasma levels of factor VIII: C increases the risk of thrombosis (control of patients at risk is necessary).

Patients with hemophilia A may develop inhibitory antibodies, while there is an inadequate clinical response to the administration of the drug.

Interaction. Do not mix with other drugs or administer at the same time using the same infusion set.

special instructions. With the introduction of the drug, it is necessary to carefully monitor the condition of patients. Early signs of a hypersensitivity reaction are urticaria, a generalized rash, chest tightness, dyspnea, hypotension, and anaphylaxis. If these symptoms occur, the administration of the drug should be stopped immediately. With the development of anaphylactic reactions, patients should be examined for the presence of inhibitory antibodies.

When using drugs derived from human blood or plasma, the possibility of transmission of infectious agents cannot be completely excluded, therefore it is recommended preventive vaccination against hepatitis A and B.

Long-term treatment of patients with von Willebrand disease with a drug containing von Willebrand factor and coagulation factor VIII can cause an excessive increase in coagulation factor VIII:C, which increases the risk of thrombosis (control of the concentration of coagulation factor VIII:C is necessary).

The risk of developing inhibitory antibodies in hemophilia A reaches a maximum during the first 20 days after the appointment, less often after the first 100 days of the drug. The possibility of using the drug in the presence of inhibitory antibodies to coagulation factor VIII has not been established.

To administer the drug, only the kit for dissolution and intravenous administration provided in the kit should be used. Other devices are able to adsorb clotting factors on their inner surface, leading to a decrease in the effectiveness of treatment.

Study Information

Special instructions: Do not conduct research during acute periods diseases and while taking anticoagulant drugs (after cancellation, at least 30 days must pass). Biomaterial for research must be taken on an empty stomach. At least 8 hours should elapse between the last meal and blood sampling.

GENERAL RULES OF PREPARATION FOR RESEARCH:

1. For most studies, it is recommended to donate blood in the morning, from 8 to 11 am, on an empty stomach (at least 8 hours should elapse between the last meal and blood sampling, water can be drunk in normal mode), on the eve of the study, a light dinner with restriction of fatty foods. For infection tests and emergency investigations, it is acceptable to donate blood 4-6 hours after the last meal.

2. ATTENTION! Special preparation rules for a number of tests: strictly on an empty stomach, after 12-14 hours of fasting, you should donate blood for gastrin-17, lipid profile (total cholesterol, HDL cholesterol, LDL cholesterol, VLDL cholesterol, triglycerides, lipoprotein (a), apolipo-proten A1, apolipoprotein B); a glucose tolerance test is performed in the morning on an empty stomach after 12-16 hours of fasting.

3. On the eve of the study (within 24 hours), exclude alcohol, intense physical activity, medication (as agreed with the doctor).

4. 1-2 hours before donating blood, refrain from smoking, do not drink juice, tea, coffee, you can drink non-carbonated water. Exclude physical stress(running, fast climbing stairs), emotional arousal. It is recommended to rest and calm down 15 minutes before donating blood.

5. You should not donate blood for laboratory testing immediately after physiotherapy, instrumental examination, X-ray and ultrasound research, massage and other medical procedures.

6. When monitoring laboratory parameters in dynamics, it is recommended to conduct repeated studies under the same conditions - in the same laboratory, donate blood at the same time of day, etc.

7. Blood for research should be donated before the start of taking medications or no earlier than 10-14 days after they are discontinued. To evaluate the control of the effectiveness of treatment with any drugs, it is necessary to conduct a study 7-14 days after the last dose of the drug.

If you are taking medication, be sure to tell your doctor about it.

GENERAL PHARMACOPEIAN AUTHORIZATION

Introduced for the first time

real monograph applies to methods for determining the activity of factors of the human blood coagulation system I, II, VII, VIII, IX, X, XI, von Willebrand factor, antithrombin III in plasma and blood products.

GENERAL PROVISIONS

Determination of the activity of coagulation factors is based on 2 approaches:

1. One step method. Restoration of the blood coagulation process in factor-deficient plasma after adding the preparation of this factor (clotting method).
2. two-stage method. In the first step, using a specific cofactor, the proteolytic activity of factor II or factor X is activated to form, respectively, activated factor IIa or Xa. At the second stage, the amount of the formed activated factor is determined by the reaction of its cleavage of a specific chromogenic peptide (chromogenic method).

It is possible to carry out the chromogenic method in 2 ways: according to the kinetics of chromogen formation under the action of an activated factor or according to the end point of chromogen accumulation for a certain incubation time.

For carrying out both methods, it is possible to use plastic test tubes, plates, optical-mechanical, mechanical semi-automatic and fully automated coagulometers.

In all methods, activity is calculated by comparing the activity of the test sample with the activity of the NIBSC International Standard or a secondary clotting factor standard calibrated against the international standard in international units of activity (IU). The equivalence of the international standard in IU is established by WHO. For 1 IU (100%) take the activity of the coagulation factor in 1.0 ml of fresh normal pooled blood plasma from 300 donors. Activity can be expressed in IU/ml, IU/vial, IU/mg of protein, and as a percentage of the amount declared by the manufacturer.

FACTORS

FactorI (fibrinogen)

Clotting method

Determination of fibrinogen activity is carried out by the Clauss method.

Method principle

When thrombin is added to a sample containing fibrinogen, fibrin clot formation is observed. When using thrombin with high activity (~10 IU/ml) and samples with low fibrinogen concentration (below 100 mg/dcl), a linear relationship can be obtained.

For the determination of fibrinogen, commercial test systems are used, which include a thrombin reagent containing a heparin inhibitor and a sample dilution buffer or a commercial thrombin reagent.

Fibrinogen concentration is expressed in mg/dcl. To construct a calibration graph, a standard fibrinogen sample or a plasma calibrator certified according to the international standard is used. A standard sample or a sample of plasma-calibrator is dissolved in distilled water according to the instructions. Prepare 5 serial dilutions of the standard sample, starting at a concentration of ~100 mg/dcl, using sample dilution buffer pH = 7.3 ± 0.1. The analysis is carried out at a temperature of 37°C according to the instructions for the kit. For each dilution, the clot formation time is determined three times. Based on the results obtained in logarithmic coordinates, a calibration graph of the dependence of the time of clot formation on the concentration of fibrinogen is built.

Prepare 2 dilutions of the test sample below 100 mg/dl. For each dilution, the clot formation time was determined three times. The amount of fibrinogen in each dilution is determined by the calibration graph.

FactorII(thrombin)

1. Clotting method

Determination of factor II activity is carried out using factor II deficient human plasma as a substrate. The coagulation process is activated by adding thromboplastin to the calcium mixture.

To dilute the standard and preparations, use NaCl-imidazole buffer pH 7.3 ± 0.1 with the addition of 0.1% bovine or human albumin. To construct a calibration graph, a series of serial dilutions of the factor II standard is prepared in the activity range from 0.3 to 1 IU/ml. The drug is diluted to a concentration of less than 1 IU / ml. Analyze 3 dilutions of the drug. For each sample, the measurement of clotting time is carried out at least 2 times. The measurement is carried out within 1 hour after dilution of the drug.

The analysis is carried out at a temperature of (37±0.5)°C. Add 50 µl of factor II deficient plasma and 50 µl of the standard or preparation dilution to a plastic tube. The mixture is incubated at (37 ± 0.5)°C for 120–240 s. The clotting time is determined from the moment of adding to the mixture of 200 μl of calcium thromboplastin preheated to a temperature of (37±0.5)°C. Depending on the technique of setting up the analysis, the volumes of reagents may vary in the appropriate proportion.

The calibration graph is built in semi-logarithmic coordinates. On the logarithmic abscissa axis, the activity values ​​of factor II are plotted; on the ordinate axis, the clotting time of the corresponding dilutions of the standard is plotted. Factor II activity for each dilution of the test sample is found from the calibration curve. FII activity ( BUT

k

1. Chromogenic method

The method is based on a comparison of the enzymatic activity of factor IIa, formed after the specific activation of factor II, in relation to a specific chromogenic peptide substrate with the same activity of a standard sample (international or similar).

Factor II is activated by the ecarin activator isolated from viper venom. Activated factor II (thrombin) selectively cleaves the chromogenic substrate (H-D-phenylalanine-L-pipecolyl-L-arginine-4-nitroanilide dihydrochloride, 4-toluenesulfonylglycyl-prolyl-L-arginine-4-nitroanilide, H-D-cyclohexylglycyl-α-aminobutyryl- L-arginine-4-nitroanilide, D-cyclohexylglycyl-L-alanyl-L-arginine-4-nitroanilide diacetate) to form P-nitroaniline. The reaction kinetics are examined photometrically at 405 nm. The value of optical density is proportional to the activity of factor II.

The determination of factor II by the chromogenic method is carried out using special test kits. The analysis is performed in accordance with the instructions for the kit. The standard sample and preparation are pre-diluted with plasma deficient in factor II to a concentration of ~ 1 IU / ml (basic dilution). From the main dilution, 3 dilutions of the standard sample and 3 dilutions of the preparation with Tris-saline buffer solution pH 8.4 are prepared. Each dilution of the standard sample is determined twice, the obtained values ​​are used to build a calibration graph. The test samples are determined three times.

The tests are carried out in manual mode using a microplate and a spectrophotometer maintaining the temperature in the range of (37 ± 0.5)°C or in automatic mode using a coagulometer.

For manual testing, 25 µl of each dilution of the test preparation or standard sample is added to the wells of the microplate. 125 µl dilution buffer, 25 µl ecarin are added to each well and incubated at (37±0.5)°C for 2 minutes. At the end of the incubation time, 25 µl of factor IIa chromogenic substrate is added to each well.

BUT/min).

If continuous absorbance measurement is not possible, determine absorbance at 405 nm at successive time intervals (e.g. 40 s) and plot absorbance versus time and calculate ∆ BUTBUT

1. Test for the absence of thrombin

For testing, a solution of the preparation restored in accordance with the instructions is prepared. If there is heparin in the preparation, it is neutralized by adding protamine sulfate at the rate of 10 μg of protamine sulfate per 1 IU of heparin.

Preparation of fibrinogen solution

0.3 g of fibrinogen is dissolved in 100 ml, mixed and incubated for 15-20 minutes at room temperature.

The shelf life of the solution is 1 month at a temperature of 2 to 8°C.

Preparation of thrombin solution

The lyophilisate is dissolved in accordance with the manufacturer's instructions, kept for 15-20 minutes at room temperature and diluted with 0.9% sodium chloride solution to a thrombin content of 1 IU/ml.

The shelf life of the solution is 6 months at a temperature of minus 20°C. The solution after thawing is not subject to re-freezing.

Definition progress

Equal volumes of the reconstituted drug and fibrinogen solution are added to 2 test tubes. Equal volumes of thrombin solution and fibrinogen solution are added to the third tube (control sample), the contents of the tubes are mixed with rotational movements. One tube with the reconstituted drug is incubated in a water bath at a temperature of 37°C for 6 hours, the other tube with the reconstituted drug is at room temperature for 24 hours. The presence or absence of coagulation (clot) is noted.

The control sample is incubated in a water bath at 37°C and note the time of clot formation.

The criterion for the acceptability of the results is coagulation in the control sample after 30 s.

FactorVII

1. Clotting method

Determination of factor VII activity is carried out using human plasma deficient in factor VII. The coagulation process is activated by adding thromboplastin to the calcium mixture.

To dilute the standard and preparations, NaCl-imidazole buffer pH 7.3 is used with the addition of a 0.1% solution of human or bovine albumin. To construct a calibration curve, a series of serial dilutions of the factor VII sample standard is prepared in the range from 0.3 to 1 IU/ml. The drug is diluted to a concentration of less than 1 IU / ml. Analyze 3 dilutions of the drug. For each sample, the measurement of clotting time is carried out at least 2 times. The measurement is carried out immediately after dilution of the drug.

The analysis is carried out at a temperature of (37±0.5)°C. 50 µl of factor VII-deficient plasma and 50 µl of the dilution of the standard or drug are added to a plastic tube. The mixture is incubated at (37±0.5)°C for 120–240 s. The clotting time is determined from the moment of adding to the mixture of 200 μl of calcium thromboplastin preheated to a temperature of (37±0.5)°C. Depending on the technique of setting up the analysis, the volumes of reagents may vary in the appropriate proportion.

The calibration graph is built in semi-logarithmic coordinates. On the logarithmic abscissa axis, the activity values ​​of factor VII are plotted, along the ordinate axis, the clotting time of the corresponding dilutions of the standard. Factor VII activity for each dilution of the test sample is found from the calibration curve. FVII activity ( BUT) in the test sample is calculated by the formula:

- activity of the corresponding dilution of the test sample, found from the calibration graph;

k- dilution of the test sample.

1. Chromogenic method

In the presence of tissue factor (TF) and Ca 2+ ions, factor VII is activated (formation of FVIIa). The complex of FVIIa, TF, Ca 2+ and a phospholipid activates factor X. Activated factor X (FXa) selectively cleaves the chromogenic substrate FXa-1 methoxycarbonyl-D-cyclohexylalanyl-glycyl-L-arginine- n-nitroanilide acetate to form P-nitroaniline. The study of the sample is carried out by the photometric method at 405 nm. The absorbance value (or increase in absorbance) is proportional to the amount of factor VII.

The determination of factor VII by the chromogenic method is carried out using special test kits. The analysis is performed in accordance with the instructions for the kit. The standard sample and preparation are pre-diluted with plasma deficient in factor VII to a concentration of ~ 1 IU / ml (basic dilution). From the main dilution, 3 dilutions of the standard sample and 3 dilutions of the preparation with tris-saline buffer pH 7.3 - 8.0 are prepared using a buffer solution with the addition of 0.1% human or bovine albumin. Each dilution of the standard sample is determined twice, the obtained values ​​are used to build a calibration graph. The test samples are determined three times.

Dilutions of the test drug or standard sample are added to the wells of the microplate, calcium-thromboplastin mixture, factor X solution are added and incubated at a temperature of (37 ± 0.5) ° C for 2 to 5 minutes, after which the factor Xa chromogenic substrate solution is added.

Measure the change in optical density at a wavelength of 405 nm, either in kinetic mode, or interrupt the hydrolysis reaction after 3-15 minutes by adding a 20% (v/v) solution of glacial acetic acid and measure the optical density.

FactorVIII

1. Clotting method

Determination of factor VIII activity is carried out using human plasma deficient in factor VIII. The source of phospholipids necessary for coagulation is the APTT reagent.

To dilute the standard and preparations, NaCl-imidazole buffer solution pH (7.3 ± 0.1) is used, with the addition of a 0.1% solution of human or bovine albumin. To build a calibration graph, a series of serial dilutions of the standard is prepared, starting from a concentration of 2 IU/ml. The drug is diluted to a concentration of ~ 0.5 - 2 IU / ml. Analyze 3 dilutions of the drug. For each sample, the measurement of clotting time is carried out at least 2 times. The measurement is carried out within 1 hour after dilution of the drug.

The analysis is carried out at a temperature of (37±0.5)°C. 100 µl of factor VIII-deficient plasma, 100 µl of the dilution of the standard or drug, and 100 µl of the APTT reagent are added to a plastic tube and incubated at a temperature of (37±0.5)°C for 2 minutes. The clotting time is fixed from the moment of adding to the mixture 100 µl of 0.025 M calcium chloride solution preheated to a temperature of (37 ± 0.5)°C. Depending on the technique of setting up the analysis, the volumes of reagents may vary in the appropriate proportion.

The calibration graph is built in semi-logarithmic coordinates. The values ​​of factor VIII activity are plotted along the logarithmic abscissa axis, and the clotting time of the corresponding dilutions of the standard is plotted along the ordinate axis. Factor VIII activity for each dilution of the test sample is found from the calibration curve. FVIII activity ( BUT) in the test sample is calculated by the formula:

- the activity of the corresponding dilution of the study drug, found from the calibration graph;

k- dilution of the test sample.

1. Chromogenic method

Quantitative determination of factor VIII is carried out using a set of reagents, in which factor VIII is a cofactor for factor IXa when factor X is activated to form Xa, which cleaves the chromogenic substrate.

Method principle

In the presence of Ca 2+ ions and phospholipids, factor X is activated into Xa by factor IXa. With an excess of factor X and optimal amounts of Ca 2+ , phospholipids, and factor IXa, the rate of factor X activation depends linearly on the amount of factor VIII. Factor Xa hydrolyzes the chromogenic substrate S-2765 (N-a-Z-DArg-Gly-Arg-pNA) to release the chromogenic group of pNA, the color of which is recorded spectrophotometrically at 405 nm. The amount of factor Xa formed, and hence the intensity of staining, is proportional to the factor VIII activity in the sample.

The factor VIII activity reagent kit is stable for the period specified by the manufacturer at a storage temperature of 2 to 8 ° C.

The reagent kit contains:

1. 7.7 mg chromogen S-2765 supplemented with synthetic thrombin inhibitor I-2581. The reagent is reconstituted in 6.0 ml of sterile water for injection. The reconstituted solution is stable for 1 month at 2 to 8°C. Before use, heated to a temperature of 37°C.
2. Bovine factors reagent: 0.3 U factor IX, 2.7 IU factor X and 1 NIH U thrombin lyophilized in the presence of 40 mmol CaCl 2 and 0.2 mmol phospholipids. The reagent is reconstituted in 2.0 ml of sterile water for injection. The reconstituted solution is stable for 12 hours at 2 to 8°C, 2 weeks at minus 30°C and 1 month at minus 80°C. Do not store at minus 20°C. Before use, heat to 37 about S.
3. Concentrate ×10 Tris buffer. Stable for 1 month at 2 to 8°C. Before use, dilute with sterile water for injection in a ratio of 1:10.

Additional reagents:

1. International Standard - human clotting factor VIII concentrate solution (NIBSC, Eur.Pharm.Ref.Std. BRP H 0920000) or plasma calibrated to the international factor VIII standard.
2. Control normal or pathological plasma calibrated to the international factor VIII standard.
3. 0.9% sodium chloride solution.
4. 20% acetic acid or 2% citric acid (used in the Chromogen Storage Endpoint Method).
5. Water laboratory deionized

Equipment:

1. Plastic test tubes;
2. microplates;
3. Thermostat 37 o C;
4. Spectrophotometer 405 nm or microplate reader 405 nm;
5. Calibrated pipettes;
6. Vortex;
7. Stopwatch.

The test sample is diluted to the expected activity of 1 IU/ml before determination.

The determination can be carried out in kinetic mode and at the end point, in test tubes (macromethod) and in microplates (micromethod).

Calibration

During each determination, a calibration curve is constructed. Dilution of a standard sample is prepared in 2 stages: preliminary dilution to an activity of 1–2 IU/ml and final dilutions to build a calibration dependence in the range of 0–2 IU/ml. After dilution, the determination should be carried out within 30 minutes.

Definition progress

Determination in test tubes

200 µl of a diluted standard, control or test sample is added to test tubes, incubated for 3-4 minutes at a temperature of 37 ° C, 50 µl of a pre-warmed factor reagent is added, incubated for 2-4 minutes at a temperature of 37 ° C and 50 µl is added chromogen solution.

Determination in microplates

Add 50 µl of a diluted standard, control or test sample to the wells of the microplate, incubate for 3-4 minutes at 37°C, add 50 µl of pre-warmed factor reagent, incubate for 2-4 minutes at a temperature of 37 ° C and add 50 µl of chromogen solution.

Kinetic method of determination

After adding the chromogen solution for 2-10 minutes, the change in the optical density of the solution is measured at 405 nm.

Definition by endpoint

After adding the chromogen solution, the mixture is continued to be incubated at a temperature of 37 ° C for 2-10 minutes, after which 50 μl of 20% acetic acid or 2% citric acid. Measure the optical density of the solution against the buffer at 405 nm.

Calculations

Plot the change in optical density per minute (for the kinetic method) or optical density (to determine the end point) of dilutions of the standard solution on the concentration of factor VIII in them. The activity in the test sample is determined from the calibration curve, taking into account the preliminary dilution of the sample.

FactorIX

1. Clotting method

Determination of factor IX activity is carried out using human plasma deficient in factor IX. The source of phospholipids necessary for coagulation is the APTT reagent.

To dilute the standard and preparations, NaCl-imidazole buffer pH 7.3 is used with the addition of a 0.1% solution of bovine or human albumin. To construct a calibration graph, a series of serial dilutions of the standard is prepared in the range from 0.3 to 1 IU/ml. The drug is diluted to a concentration of less than 1 IU / ml. Analyze 3 dilutions of the drug. For each sample, the measurement of clotting time is carried out at least 2 times. The measurement is carried out within 1 hour after dilution of the drug.

The analysis is carried out at a temperature of (37±0.5)°C. 100 µl of factor IX-deficient plasma, 100 µl of the standard or drug dilution, and 100 µl of the APTT reagent are added to a plastic tube and incubated at (37 ± 0.5)°C for 2 minutes. The clotting time is fixed from the moment of adding 100 µl of 0.025 M calcium chloride solution preheated to a temperature of (37 ± 0.5) °C to the mixture. Depending on the technique of setting up the analysis, the volumes of reagents may vary in the appropriate proportion.

The calibration graph is built in semi-logarithmic coordinates. On the logarithmic abscissa axis, the activity values ​​of factor IX are plotted; on the ordinate axis, the clotting time of the corresponding dilutions of the standard is plotted. Factor IX activity for each dilution of the test sample is found from the calibration curve. FIX activity ( BUT) in the test sample is calculated by the formula:

- activity of the corresponding dilution of the test sample, found from the calibration graph;

k- dilution of the test sample.

FactorX

1. Clotting method

Determination of factor X activity is carried out using factor X deficient human plasma. The coagulation process is activated by adding thromboplastin to the calcium mixture.

To dilute the standard and preparations, use NaCl-imidazole buffer solution pH 7.3 with the addition of 0.1% solution of human or bovine albumin. To build a calibration graph, prepare a series of serial dilutions of the factor X standard in the range from 0.3 to 1 IU/ml. The drug is diluted to a concentration of less than 1 IU / ml. Analyze 3 dilutions of the drug. For each sample, the measurement of clotting time is carried out at least 2 times. The measurement is carried out immediately after dilution of the drug.

The analysis is carried out at a temperature of (37±0.5)°C. Add 50 µl of Factor X-deficient plasma and 50 µl of the standard or preparation dilution to a plastic tube. The mixture is incubated at (37±0.5)°C for 120–240 s. The clotting time is determined from the moment of adding to the mixture of 200 μl of calcium thromboplastin preheated to a temperature of (37±0.5)°C. Depending on the technique of setting up the analysis, the volumes of reagents may vary in the appropriate proportion.

The calibration graph is built in semi-logarithmic coordinates. The logarithmic abscissa axis plots the activity values ​​of factor X , along the y-axis, the clotting time of the corresponding dilutions of the standard. Factor X activity for each dilution of the test sample is found from the calibration curve. Factor X activity ( BUT) in the test sample is calculated by the formula:

- activity of the corresponding dilution of the test sample, found from the calibration graph;

k- dilution of the test sample.

1. Chromogenic method

Factor X is activated with the help obtained from snake venom FX activator. Activated factor X (FXa) selectively cleaves the chromogenic substrate FXa-1 N-α-benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-4-nitroanilide dihydrochloride, N-benzoyl-L-isoleucyl-L-glutamyl-glycyl- L-arginine-4-nitroanilide hydrochloride, methanesulfonyl-D-leucyl-glycyl-L-arginine-4-nitroanilide, methoxycarbonyl-D-cyclohexylalanyl-glycyl-L-arginine-4-nitroanilide acetate to form P-nitroaniline. Samples are examined photometrically at 405 nm. The amount of factor X is proportional to the increase in the optical density of the solution.

Determination of factor X by the chromogenic method is carried out using special test kits. The analysis is performed in accordance with the instructions for the kit. The standard sample and preparation are pre-diluted with factor X-deficient plasma to a concentration of ~ 1 IU/ml (basic dilution). From the main dilution, 3 dilutions of the standard sample (according to the instructions) and 3 dilutions of the drug are prepared using a buffer solution. Each dilution of the standard sample is determined twice, the obtained values ​​are used to build a calibration graph. The test samples are determined three times.

The tests are carried out manually using plastic tubes or microplates at a temperature of (37±0.5)°C or automatically using a coagulometer.

For manual tests, 12.5 µl of each dilution of the test drug or standard sample is added to the wells of the microplate, 25 µl of specific factor X activator from the venom of Russell's viper is added to each well and incubated at a temperature of (37 ± 0.5) ° C for 90 s, after which 150 µl of the working dilution of factor X chromogenic substrate is added to each well.

Determine the rate of change in absorbance at a wavelength of 405 nm continuously for 3 min and calculate the average rate of change in absorbance (∆ BUT/min) or measure the absorbance at a wavelength of 405 nm at successive time intervals (for example, after 30 s) and plot the absorbance versus time and calculate ∆ BUT/min as the angle of inclination of the straight line. From the values ​​∆ BUT/min of each individual dilution of the standard sample and the test drug, the activity of the test drug is calculated.

Willebrand factor

Determination of the activity of the von Willebrand factor is carried out by the method of agglutination or enzyme immunoassay.

The activity of the von Willebrand factor is determined by comparing its activity with the activity of a standard sample.

Agglutination Method

The method is based on the determination of the coenzyme activity of the von Willebrand factor during agglutination of a platelet suspension in the presence of ristocetin A.

The test may be carried out quantitatively using automated equipment or semi-quantitatively by visual assessment of agglutination in a series of dilutions.

Semi-quantitative method

Serial dilutions in 0.9% sodium chloride solution with 1–5% human albumin solution are prepared from a standard sample and a reconstituted solution of the drug to the expected content of von Willebrand factor of 0.5, 1.0 and 2.0 IU/ml. 0.05 ml of each dilution of the standard sample and the test preparation is applied to a glass slide, 0.1 ml of platelet suspension with ristocetin is added and mixed for 1 min. The dilution solution is used as a negative control. After 1 min incubation at room temperature, platelet agglutination is visually assessed. The maximum dilution at which platelet agglutination occurs is the titer of the ristocetin coenzyme activity of the sample.

quantitative method

Prepare at least 2 series of serial dilutions of the reference and test samples with dilution buffer to the expected von Willebrand factor content of 0.5, 1.0 and 2.0 IU/ml.

The determination is carried out in accordance with the instructions of the manufacturer of the automated equipment. Get the values ​​of the dependence of the change in optical absorption (degree of turbidity of the solution) from the activity of the von Willebrand factor.

Determination of the content of the von Willebrand factor in the test preparation is carried out using the coefficients of the linear equation of the dependence of the optical density of the solution on the content of the von Willebrand factor in the standard sample.

ELISA method

The method is based on the determination of the collagen-binding activity of the von Willebrand factor. Upon specific binding of von Willebrand factor to collagen fibrils and subsequent binding of enzyme-conjugated polyclonal antibodies to von Willebrand factor, a chromophore is formed after the addition of a chromogenic substrate, which can be quantified by spectrophotometric method. Exist linear dependence between collagen binding to von Willebrand factor and optical density.

The determination is carried out using test systems approved for use in healthcare practice in Russia in accordance with the instructions for use.

For testing, prepare at least 3 consecutive series of dilutions of the standard and test samples using dilution buffer to the expected content of von Willebrand factor of 1.0 IU/ml. Next, tests are carried out in accordance with the manufacturer's instructions for the test system used.

Activated clotting factors blood

The determination is carried out by the coagulometric method.

For testing, a reconstituted solution of the drug is prepared. If there is heparin in the preparation, it is neutralized by adding protamine sulfate at the rate of 10 μg of protamine sulfate per 1 IU of heparin. Prepare dilutions of the drug 1:10 and 1:100 using Tris buffer solution pH 7.5.

0.1 ml of standard human plasma and 0.1 ml of phospholipid solution are added to 3 test tubes, placed in a water bath with a temperature of (37 ± 0.5) ° C for 60 s, after which 0.1 ml is added to the first test tube Tris buffer solution (control sample), in the second - 0.1 ml of the test drug at a dilution of 1:10, in the third test tube - 0.1 ml of the test drug at a dilution of 1:100. Further, 0.1 ml of a 3.7 g/l calcium chloride solution heated to a temperature of 37 ° C is immediately added to the contents of each test tube. The time of clot formation from the moment of adding calcium chloride solution is noted.

The criterion for the acceptability of the results is the coagulation time in the control sample in the range from 200 to 350 s.

Specific specific activity

The specific activity of coagulation factors is calculated by the formula:

Determination of antithrombin activityIII

Chromogenic method

The method for determining ATIII activity is based on its ability to neutralize thrombin in the presence of heparin. Excess amounts of heparin and thrombin are added to the sample containing ATIII. The resulting ATIII-heparin complex neutralizes the amount of thrombin proportional to the amount of ATIII. The remaining thrombin selectively cleaves the chromogenic substrate to form P-nitroaniline, the absorption of which is determined at 405 nm. Thus, the amount of ATIII is inversely proportional to the absorption of free P-nitroaniline in the sample.

Determination of ATIII activity is carried out using commercial test systems. To build a calibration graph, use a standard ATIII sample or a plasma calibrator certified according to the international standard. The standard sample or plasma calibrator is dissolved in distilled water according to the instructions in the instructions. The dependence of the optical density on the activity of ATIII is linear in the range of ATIII activity from 0.1 to 1.0 IU/ml. Using heparin buffer, prepare 4 dilutions of a standard or calibrator plasma with an ATIII activity of 0.1 to 1.0 IU/mL. The analysis is carried out at a temperature of 37°C according to the scheme given in the kit instructions. For each dilution, the optical density value at 405 nm is determined three times and a calibration plot of absorbance versus ATIII activity is plotted in linear coordinates.

Prepare 2 dilutions of the test sample with an estimated ATIII activity of less than 1.0 IU/ml. The determination of ATIII activity in the test samples is carried out at a temperature of 37°C according to the instructions in the instructions for the kit. The activity of ATIII in the tested dilutions is found from the calibration curve. The activity of ATIII in the test sample is defined as:

A x– ATIII activity in the appropriate dilution;

k- dilution of the sample.

Quantification of heparin

1. Clotting method

The method is based on the ability of heparin to prolong the clotting time of normal plasma due to the inhibition of a number of factors.

For the analysis, normal human plasma, a standard sample of heparin, an APTT reagent and a solution of calcium chloride 0.025 M are used. A 0.9% sodium chloride solution is used as a diluent for the standard and test samples. A standard sample of heparin is dissolved in distilled water according to the instructions in the instructions. Prepare 3 dilutions of a standard sample with a heparin activity of 0.3, 0.4 and 0.5 IU/ml. Standard samples with these activities should prolong the clotting time of normal plasma by at least 1.5 times, otherwise dilutions with higher heparin activity should be used. In parallel, 3 dilutions of the test sample are prepared so that approximately the activity of heparin in these dilutions falls within the range of heparin activity in dilutions of the standard sample.

The analysis is carried out using an automatic or semi-automatic coagulometer in plastic test tubes at a temperature of 37°C. 100 µl of normal human plasma, 100 µl of a dilution of a standard or test sample, or 100 µl of 0.9% sodium chloride solution (blank experiment) are added to the tube, 100 µl of the APTT reagent are added and the mixture is incubated for 120–240 s at a temperature ( 37±0.1)°С. Then, 100 μl of 0.025 M calcium chloride solution preheated to a temperature of 37°C is added to the test tube and the clotting time of the sample is recorded. Depending on the technique of setting up the analysis, the volumes of reagents can be changed in proportion. The clotting time of normal plasma (blank) should be 25–40 s. For each dilution of standard and test samples, the clotting time is determined three times.

1. Chromogenic method

The method is based on the cleavage of a chromogenic substrate specific for activated factor X (FXa). When added to a sample containing heparin, excess quantities ATIII and FXa inhibit the amount of FXa proportional to the amount of heparin. The remaining FXa is cleaved from a specific chromogenic substrate P-nitroaniline, the absorption of which is determined at 405 nm. Thus, the amount of absorption is inversely proportional to the amount of heparin. This method determines the content of both unfractionated and low molecular weight heparin in anti-Xa units.

The amount of heparin is determined by the chromogenic method using commercial test systems. To construct a calibration graph, a standard heparin sample or a plasma calibrator certified according to the international standard is used. The standard sample or plasma calibrator is diluted in distilled water according to the instructions. Prepare 4 dilutions of a standard with a heparin concentration of less than 1 anti-Xa U/mL using dilution buffer pH 8.4. The analysis is carried out at a temperature of 37°C in accordance with the instructions for the kit. For each dilution, the absorbance at 405 nm is determined three times, after which a calibration graph of the dependence of the absorbance on the heparin concentration is plotted in linear coordinates. The dependence is linear in the range of heparin concentrations 0 – 1.0 anti-Xa units/ml.

Prepare 2 dilutions for the test sample with a heparin concentration of less than 1 anti-Xa U/mL. The analysis is carried out according to the instructions for the set at a temperature of 37°C. For each dilution, the absorbance value is determined three times.

The determination is carried out manually using plastic test tubes, microplates or automatically using a coagulometer.

For testing in manual mode, 20 µl of standard human plasma and 20 µl of antithrombin III solution are added to the microplate wells. Further, 20, 60, 100 and 140 μl of the test or standard preparation are added to these wells, respectively, and the solution volume in each well is adjusted to 200 μl with buffer (heparin activity in the final reaction mixture is 0.02 - 0.08 IU / ml).

40 µl from each well of the plate is transferred to the second series of wells, into which 20 µl of the bovine factor Xa solution are added and incubated at a temperature of (37 ± 0.5) ° C for 30 s, after which 40 µl of the solution are added to the wells factor Xa chromogenic substrate and incubated again at (37 ± 0.5)°C for 3-15 min, measuring the rate of degradation of the substrate by continuous measurement of optical density at a wavelength of 405 nm (kinetic mode) or after stopping the reaction by adding 20% (v/v) glacial acetic acid solution (final point of determination).

When conducting research in automatic mode using a coagulometer, the values ​​of the dependence of the change in optical density on the concentration of heparin are obtained.