Features of mass screening of newborns for cystic fibrosis. Conducting neonatal screening of newborns in the maternity hospital for hereditary diseases: a blood test from the heel

Screening for cystic fibrosis (cystic fibrosis) is the only way to find out about the abnormal development of a protein responsible for the correct transport of chloride ions to organs that secrete various secrets: sweat, mucus, saliva, digestive juices and fluids necessary for conception of offspring even at the asymptomatic stage this autosomal recessive hereditary disease.

Despite the fact that this disease is associated with a serious violation of the functions of the most important body systems, such as the pancreas, intestines, bronchi and lungs, liver, excretory and genital organs, many mutations in the CFTR gene cause various symptomatic forms that do not necessarily manifest themselves in childhood and adolescence. So, if the patient has a mild form of a genetic pathology, the characteristic signs may be either not pronounced and similar to other diseases, or absent altogether.

In addition, on this method examination should pay attention to people with aggravated heredity, who had relatives in the family who suffered from chronic ailments diagnosed as bronchial asthma, intestinal obstruction, infertility, pancreatitis and cirrhosis of the liver, as well as those who did not live up to 40 years. After all, as practice shows, patients with monogenic CFTR pathology do not live up to 35-40 years. Passing analyzes and special tests is important for such people because they may be carriers of an unfavorable mutational gene, which increases the risk of its transmission to subsequent generations.

Compulsory Cystic Fibrosis Control Program

Today, screening for cystic fibrosis in the maternity hospital is a mandatory procedure included in the complex of federal measures to combat genetic pathologies. And new parents, for sure, are familiar with the test for IRT / DNA, when a drop of blood is taken from the baby's heel, which is dried, after which doctors determine the level of the enzyme immunoreactive trypsin.

If the first stage of the examination, which is usually carried out on the 4-5th day of the baby's stay in the obstetric institution, showed a negative result, that is, the amount of IRT does not exceed the norm of 65-70 ng/ml, the child is considered to be conditionally healthy.

Screening for cystic fibrosis in newborns, in which the pancreatic enzyme level is 5-10 times higher than normal, goes to the second stage, which is carried out strictly from 21 to 28 days of a small patient's life. At this stage, repeated blood sampling and its testing for the quantitative content of IRT are performed. The normative indicator in healthy children of this period of life is no more than 40 ng / mg. If, after this, the analyzes of your child confirm the activity of immunoreactive trypsin, doctors refer the parents with the baby to take sweat samples. This is necessary in order to either confirm or exclude a monogenic mutation, since an increase in the level of RTI in babies can be the result of different pathologies, among which:

• fetal hypoxia;
• intrauterine infections;
• conjugative jaundice;
• perinatal stress;
• chromosomal rearrangements.

To date, sweat testing alone is the most accurate way to diagnose cystic fibrosis in infants and children under 2 years of age. Moreover, they must be carried out at least 2-3 times, since the indicators in different periods of testing may differ.

Differences between neonatal and prenatal diagnosis

These two methods for detecting a mutational gene are fundamentally different from each other in that prenatal examination can detect the disease even at the stage of fetal development, and neonatal screening for cystic fibrosis involves the detection of a potential danger after the baby is born in the first months of his life.

In addition, such diagnostic methods operate with various types of analyzes. At prenatal diagnosis the saliva and blood of the parents are examined, and a chorionic biopsy and amniopuncture are also performed.

During the implementation of the NS for cystic fibrosis, specialists from various laboratories and medical centers focus on various research tactics. For example, in Europe and the United States, the analysis of RTI is considered ineffective in the context of numerous mixing of a large number of nationalities. Therefore, they use an alternative method - the detection of pancreatic protein (PAP), which can be isolated or combined with increased level pancreatic enzyme. On the basis of this technique, physicians have developed a special kit for assessing PAP + IRT, which is successfully used by doctors in leading Russian clinics.

Many parents ask themselves the question: “What should I do if the HC has confirmed the presence of cystic fibrosis?”. Experienced specialists recommend immediately contacting highly specialized doctors with rich experience in treating monogenic mutations. This will reduce the period of psychological adaptation of newly-made parents to the state of their baby and provide professional assistance in a short time. If an exacerbation of pathology does not occur in an infant, it is enough for parents to see a doctor every two weeks during the first three months of a child's life. From a year on, visits to a specialist will become less frequent - once a quarter. Most importantly, in the period up to 2 years, when the risk of exacerbations of symptoms is greatest, carefully monitor the health of your child and adhere to effective strategy treatment prescribed by a pediatrician.

Neonatal screening (NS) for serious disease cystic fibrosis (CF) was already carried out in Europe in the early 70s of the last century, but these were only the first attempts. These studies included the analysis of meconium for the content of albumin in it. In 1979, in the blood plasma of newborns, they learned to determine the level of immunoreactive trypsin (IRT), which increases with cystic fibrosis. This event was the starting point for further research in the conduct of mass screening of newborns for the presence of cystic fibrosis.
When the first cloning of the CFTR gene was carried out in 1989, the possibilities of HC expanded dramatically. It has become possible to include DNA analysis in CF screening protocols.

Data on neonatal screening in the world

In Europe, a survey was conducted in 1.6 million newborns, of which 400 babies were identified who had symptoms of cystic fibrosis.

In 2008, the number of children screened almost doubled. This increase is due to the introduction of NS in the UK and Russia. This program has fully justified itself not only in the medical component, but also in the economic one.

With the possibility of early detection of the disease, treatment can be started earlier, which subsequently leads to an improvement in the quality of life of patients and the prognosis of the disease. The introduction of HC and genotyping for the CFTR gene has led to the possibility of early family planning, taking into account the burdened gene pool.

NS options

In European countries, there are about 26 variants of the National Assembly, which consist of 2-4 stages. The first stage everywhere is the measurement of the level of immunoreactive trypsin in the blood in the first week of a newborn's life. The sign is very sensitive, but its specificity is insufficient, since an increase in IRT also occurs with conjugative jaundice, perinatal stress, intestinal atresia, and renal failure. Moreover, IRT levels are increased in North Americans and African Americans compared to Europeans. Taking blood for cystic fibrosis

The second stage is necessary to increase specificity. It is not possible in a society where a huge number of nationalities exist together to identify patients with mutated genes by determining RTI/DNA.

Another alternative method of the second stage is to find the pancreatitis-associated protein (PAP) in a single form or in combination with RTI. This approach can help avoid problems associated with the detection and analysis of CFTR mutations. Currently, there is a developed combined method: a set for the definition and evaluation of PAP+IRT. Research is being planned.

The above programs need to be combined and they should be used in relatives with aggravated pathology and in the general population, because brothers and sisters of patients with cystic fibrosis can contain a recessive gene in half of the cases, that is, be carriers.
Negative aspects of the National Assembly

Upon receipt of a positive result for CF, immediate therapy should be carried out. It is during the first period of awareness that parents can worry and have great doubts about the presence of CF in their child. If there is little time between the obtained screening data and the final diagnostic confirmation, this situation has a psychologically favorable effect on the condition of the parents of a small patient, which contributes to the early start of adequate therapy and the development of a trusting relationship between them and the doctor.

These relationships are often hard to establish and sometimes impossible. With NS, there is a possibility of false positive tests. Thus, the task of scientists is to determine the smallest possible percentage of false positive tests.

Protocol of the National Assembly in Russia

1. IRT 2;
2. sweat test;
3. DNA diagnostics.

Since 2007, NS on CF has been introduced as a mandatory measure for the detection of severe hereditary diseases, which include phenylketonuria, hypothyroidism, galactosemia, and adrenogenital syndrome.

The cost of analysis in Russia is high (about $ 100), so NA is performed quite rarely.

sweat test

Mainly in medical centers Europeans carry out sweat tests for the presence of chlorides. In Russia, two systems are registered for the determination of chlorides in sweat fluid. This is an indirect method for the determination of these substances.

Sweat collection and analysis systems

The Macroduct system with an American-made Sweat-Chek sweat analyzer is successfully used in children in the first months of life. The analysis can be carried out with its help outside the laboratory for 30 minutes.

The Nanoduct device is also used, which is equipped with a system for stimulating sweating by electrophoresis of a 0.1% pilocarpine solution and a sweat conductance analyzer.

For analysis, 3 to 6 µl of sweat is needed. Therefore, this apparatus is very widely used as a technical equipment for mass screening. Results of 80 mmol/l are considered positive. Borderline indicators - 60-80 mmol / l.

Survey data

Over three years of research, more than 4 million newborns underwent NS for cystic fibrosis in children. Of all the examined, 416 babies with signs of CF were identified. Thus, the frequency of occurrence in Russia is 1:10,000 newborns.

Repeated studies are often not carried out for children with positive tests (with an increased level of RTI), because parents refuse further studies.

Clinical examination of newborns

When a pathology is detected, babies are observed by doctors every 2 weeks for 3 months, then every month for the next six months, after which every 2 months until 1 year, and from a year every quarter.

It is important to monitor patients without any manifestations of the disease. A scatological examination is carried out every month up to 1 year, pancreatic elastase is determined twice in the first year of life, a complete blood count. With the development of an exacerbation of the pathological process, a deeper and more thorough examination is necessary.

Cystic fibrosis treatment

Therapeutic measures begin from the moment the disease is diagnosed. The amount of therapy will depend on the clinical manifestations and the breadth of organ damage. In the vast majority of patients, all symptoms occur in the 1st year of life and in the 1st month of life.

For newborns, kinesiotherapy is used using massage, vibration, stroking, and ball exercises. All activities should be enjoyable for the baby.

When attaching bronchial obstruction, bronchodilators and mucolytics are indicated.

If there are manifestations of dyspepsia, enzymes are prescribed as a replacement treatment and fat-soluble vitamins.

Conclusion

Rate value neonatal screening for cystic fibrosis in Russia will be possible after a few years. At the same time, the state should understand the importance of these events and in every possible way improve the conditions for their implementation.

Cystic fibrosis in newborns is a dangerous pathology that can cause serious harm to the body. The disease affects the organs that are necessary for the production of certain enzymes. Quite often, with such a diagnosis in infants, the glands that must produce sweat and mucus suffer. In this case, the resulting enzymes have too high a viscosity, so they quickly thicken. Against this background, the removal of some components from the body becomes difficult. Diagnosis of cystic fibrosis in newborns is based on screening. Additionally, the doctor takes a sweat sample. Only after this, a course of treatment is prescribed, which will minimize the likelihood of anxiety symptoms.

Symptoms of the disease

According to statistics, about 20% of all babies suffer from intestinal obstruction after birth. In this case, meconium ileus is diagnosed. The disease develops in case of insufficient entry into the organs of the digestive system of sodium, chlorine and water. In the process of its progression, the stomach and intestines suffer. At the next stage, meconium is blocked in them. This feces begins to form immediately after the baby is born. However, only in rare cases, this disease signals the presence of cystic fibrosis. It can only be confirmed by a specially conducted screening.

For example, jaundice is characteristic of only half of the children whose intestines suffer from obstruction. However this symptom It can also indicate the development of a more serious illness in the body. Pathology is formed against the background of excessive thickening of bile. It cannot be excreted from the gallbladder for a long time.

Additionally, there may also be the following symptoms cystic fibrosis:

• Immediately after childbirth, the baby begins to suffer from a strong cough, which greatly exhausts him.
• A detailed study of the glands located on the surface, you can find the appearance of a large amount of mucus. Its excessive accumulation appears in the bronchi. It should be removed in time. Otherwise, the risk of blockage increases.
• The screening reveals a clinical picture that confirms the presence of serious obstacles to the optimal respiratory regimen.

When mucus stagnates in the body in infants, harmful microorganisms begin to actively grow and multiply. Against this background, the risk of developing inflammation of a purulent nature increases. The child also suffers from bronchial tubes from pulmonary infection. A detailed examination can reveal a large number of violations in the work of all respiratory organs. The bronchial type of the disease is formed with a genetic predisposition to it. However, there are other reasons that significantly aggravate the situation.

Treatment for cystic fibrosis is lifelong.

Additional signs of the disease

Signs of cystic fibrosis can be seen in the analysis physical development child. For example, he is not gaining weight well. Violations lead to regression of the fiber that is under the skin.

Symptoms are easy to see with the naked eye - the child is severely stunted when compared with peers. When the disease progresses to chronic form an unpleasant odor begins to emanate from the child.

The stool becomes oily. In it you can see the remnants of food that has not yet been digested. It contains a large amount of oil impurities. That is why feces are very difficult to wash off diapers. This manifestation develops due to a malfunction of the pancreas. Clots of enzymes clog its individual parts. They are necessary for the proper breakdown of food that has entered the intestines. At the last stage of the development of the disease, the process of digestion becomes completely disturbed. Organs cannot exchange fats or proteins. To make a diagnosis, a special analysis and screening of the general health of a small patient is carried out. Additionally, a sweat test is also required.

In the presence of this diagnosis, one should understand the main points that characterize this species pathologies:

• If the child does not undergo corrective treatment, then in the future he will lag far behind his peers in development.
• Enzymes cannot enter the intestines. They begin to actively accumulate, which leads to disturbances in the work of this body.
• Within a month, pancreatic tissue is completely replaced by connective tissue. In this case, the child is diagnosed with cystic fibrosis.

With the active development of pathology, normal functioning is impossible gastrointestinal tract. Mycoviscidosis most often develops in the intestines. However, against the background of pathology, the work of the lungs is aggravated. Screening helps diagnose the disease.

Features of the diagnosis

The disease must be detected as soon as possible. Most often, neonatologists diagnose it immediately after birth. It is also important to analyze the general health of the child. Pathology can negatively affect blood vessels or other internal organs.

Diagnostic measures in without fail should be carried out within the first month of a child's life. In addition, it should be noted that a large amount of hormones accumulate in the blood of such children. They can exceed ten times the normal level. This makes it possible to obtain accurate screening results. To obtain a complete clinical picture, it is important to carry out the following diagnostic measures:

• If the child has suspicions of the presence of this pathology, then sweat samples are taken first. On their basis, it is possible to confirm or refute the diagnosis with confidence. A study is made in the ratio of chlorides to the total mass of the liquid. For the analysis, a special marker is used - pilocarpine. It is incorporated into skin through electrophoresis. Thanks to this, it is possible to activate the work of the sweat gland. After receiving sweat, it will need to be weighed and split into sodium and chlorine ions. For an accurate analysis, you will additionally need to conduct several such fences. Only after that it is advisable to proceed to screening.
• Of no small importance is the identification of problems in the work of the pancreas. It will be possible to choose the right course of treatment only on the basis of coprological screening. At the same time, the amount of fat masses in the common stool is revealed. To date, the identification of elastase-1 is most commonly used. This enzyme is produced only by the pancreas.

If cystic fibrosis has been diagnosed, then it will be necessary to establish the degree of its severity. To do this, all the results obtained are summed up and summarized in prenatal diagnostics.

Features of restoring the functioning of the body

Only combined treatment helps to get rid of cystic fibrosis. It will have to pass until the end of life. The action is aimed at liquefying sputum, followed by excretion from the bronchi. Medicines also counteract the active growth and development of bacteria harmful to the body in the lung area. The missing pancreatic enzymes are also completely replaced by drugs. Equally important is the regular intake of vitamins and minerals. With their help, it is possible to dilute bile.

Conducting a diagnostic examination

In medical practice, there are situations when it is required to prescribe a dose of the drug that is several times higher than the standard one. This need arises in the event of a violation of absorption. Additionally, it is also required to take drugs to replace the lack of iron in the body. Thanks to this, it is possible to minimize the negative impact of pathologies.

In the presence of active growth and development of viruses in the lungs, antibiotics are required. Preliminary sputum culture helps to choose the right medicine. Thanks to this, it is possible to determine the microorganism that causes most of the negative manifestations.

To adjust the course, sowing will have to be done every three months. It is best to conduct a study when the disease is not in the acute stage. If you follow this cycle, you will be able to identify potentially dangerous microorganisms even before the stage of exacerbation. The course of treatment lasts at least three weeks.

To thin sputum, it is advisable to use various mucolytics.

However, there are a few things to keep in mind when choosing them:

• If the pathology was diagnosed earlier, then it is advisable to carry out further treatment with the help of Pulmosia. Its positive impact is several times higher than that expected from the use of conventional means.
• Mucolytics are allowed to be used not only as inhalation. They can be given to children in the form of tablets. However, before the appointment without fail, the patient undergoes screening.

The disease is diagnosed in the first days of a child's life.

For quick and effective recovery of the child's body, kinesitherapy is also prescribed. It involves the regular conduct of specially selected exercises that are performed in a mask. To obtain a positive result, they should be repeated every day throughout life. The duration of the lesson directly depends on the general condition of the small patient. On average, it will take about an hour to train.

It is necessary to choose the correct remedial measure. This is possible only after confirmation of the diagnosis. To date, special centers are popular in which children undergo rehabilitation for cystic fibrosis.

Key points in feeding a child

If the baby was previously diagnosed with this, then it is best for the woman to breastfeed him. Mixtures can only exacerbate the problem. However, if there is no other option, then only a specialist in this field can choose it correctly. The process is based on screening.

When diagnosed with cystic fibrosis, a child should receive at least 120% of the nutritional norm for a child at that age. Of these, only 30% is given to fatty foods.

If the baby was prescribed classic enzymes for the pancreas, then his diet will continue to be carried out as usual. It is important to consider age characteristics.

Immediately after birth, the baby still does not know how to swallow capsules. It is allowed to pour their contents into a spoon, and then stir with milk. You can also replace it with a mixture or natural juice. The crumbs should receive tablets immediately before the meal itself. After the teeth appear, parents should ensure that the child does not chew on individual pellets.

Cystic fibrosis is a serious diagnosis. It is important to put it on time and direct all efforts to eliminate it. Parents should regularly visit the specialist's office and follow all his recommendations. To prolong the life of the crumbs and his health, he is constantly under close scrutiny.

The main goal of genetic screening is to identify in a population of people with a certain genotype that either causes a disease, or predisposes to its occurrence, or can cause a disease in offspring. The basic principles of genetic screening were developed in the 60s. of the last century, when it began to be used to detect phenylketonuria among newborns. In 1968, a group of WHO experts, following the results of screening for phenylketonuria conducted in several countries of the world, published general requirements for screening programs for newborns for hereditary diseases metabolism. These requirements are still valid today.

Basic principles of genetic screening
To general requirements implementation of newborn screening programs for hereditary metabolic diseases include the following criteria:
the frequency of the disease in the population must be sufficiently high (this requirement is not very strict, since it is related only to the cost-effectiveness of the program);
the disease should be well studied clinically and laboratory;
the disease must be severe or even fatal so that the benefit of the screening program outweighs the cost of running it;
laboratory tests should not give false negative results so as not to miss a single patient; the false-positive rate should also not be high, so as not to reduce the cost-effectiveness of the program;
laboratory tests should be simple, safe and ethically acceptable;
effective treatment for screened diseases should be developed;
the period of time from birth when the treatment gives a positive result must be precisely established;
screening should be cost effective.

Based on these requirements, neonatal screening is a system of measures, the main of which are the identification of newborns with certain diseases at the preclinical stage; early pathogenetic treatment, which makes it possible to give society full-fledged individuals; medical genetic counseling of the family, aimed at preventing the birth of a second sick child.

Screening for the most common and severe hereditary diseases falls into the category of high priority among other health problems, as it affects the motivation of the population, reduces a significant proportion of disability and provides resource savings. Neonatal screening - in principle new approach to prevention, proposed by medical genetics to practical public health.

The above requirements are met by a number of hereditary metabolic diseases. In Russia, screening of newborns for phenylketonuria has been carried out since 1985, for congenital hypothyroidism - since 1993; within the framework of the National project "Health of the Nation" since 2006, screening has been supplemented with three more diseases - galactosemia, adrenogenital syndrome and cystic fibrosis.

Phenylketonuria is a hereditary disease with an autosomal recessive type of inheritance (patients accumulate in the family in one generation).

The average frequency of phenylketonuria in European countries is 1:10,000 newborns, in the European part of Russia - 1:6500-1:7000. The main diagnostic criterion for all forms of phenylketonuria is an increased concentration of phenylalanine in the blood. The heterogeneous group of hyperphenylalaninemias includes a number of hereditary disorders of the metabolism of the amino acid phenylalanine, which result in the accumulation of this amino acid and its derivatives in biological fluids. The most common disorder is the classic form of phenylketonuria, caused by mutations in the phenylalanine hydroxylase gene on chromosome 12 (12q22-q24.2). To date, many hundreds of mutations have been identified in the phenylalanine hydroxylase gene, 8 of which are the most common. The major mutation occurring at a frequency of 45% is R408Q. As a result of a mutation in the gene, the enzyme is defective, phenylalanine cannot be converted to tyrosine and accumulates in the blood.

A metabolic block occurs, as a result of which the level of phenylalanine constantly grows and reaches such concentrations at which it becomes toxic, primarily for the developing brain of a child. Without treatment, 95% of children with phenylketonuria develop severe mental retardation, delayed motor development, convulsions, eczema on the skin, and at an older age gross behavioral disorders join.

If treatment is started early and carried out carefully, the child will not show clinical symptoms of phenylketonuria and he will grow up healthy, practically no different from his peers. The meaning of the treatment is to reduce the content of phenylalanine in the food that the child receives. This is usually achieved through special mixtures and diets. The content of phenylalanine in the blood is constantly monitored in the child and, depending on laboratory parameters, the composition of those products that will not increase the level of phenylalanine, but will ensure the normal growth and development of the child, is adjusted. A family in which there is a patient with phenylketonuria should receive medical genetic counseling, and prenatal DNA diagnostics can be performed in subsequent pregnancies.

Congenital hypothyroidism is manifested by serious violations of the growth and development of the child from birth and is caused by a complete or partial dysfunction of the thyroid gland that produces iodine-containing hormones. In most cases, congenital hypothyroidism occurs due to the absence of the thyroid gland, or its underdevelopment, or incorrect position. If congenital hypothyroidism is left untreated, the child's growth drastically slows down, severe irreversible mental retardation develops, and others develop. Clinical signs diseases. The disease progresses all the time and can lead to lifelong disability. However, if treatment is started within the first month after
birth, in the vast majority of cases, the child develops normally. Approximately 80-85% of cases of congenital hypothyroidism are non-hereditary, occur by chance, and are usually caused by a thyroid disorder whose cause is unknown. In the pathogenesis of the development of transient disorders of the functions of the pituitary-thyroid system, both an increase in the activity of the pituitary gland with an increase in the synthesis of thyroid-stimulating hormone, and the inhibition of the production of thyroid hormones can be of primary importance.

The risk group for the occurrence of transient changes, accompanied by a decrease in the concentration of thyroid hormones, include:
newborns born from mothers with complicated pregnancy, especially with fetoplacental insufficiency;
newborns born from mothers with endocrine pathology, especially with thyroid diseases;
newborns with functional immaturity due to prematurity or intrauterine malnutrition.

In 15-20% of cases, congenital hypothyroidism is inherited, usually in an autosomal recessive manner. At least 7 genes are known to mutate to cause hypothyroidism. That is why molecular genetic analysis in congenital hypothyroidism is complex and not always effective. However, since congenital hypothyroidism, both hereditary and non-hereditary, is well treated when detected early, there is little need for such genetic analysis.

Congenital hypothyroidism occurs throughout the world with approximately the same frequency - 1:3000-1:4000 newborns. The same frequency of congenital hypothyroidism in Russia. In girls, for unknown reasons, it is found twice as often as in boys. The screening program uses thyroid stimulating hormone as the primary test in dried blood spot samples. In cases with an increased content of thyroid-stimulating hormone in blood samples, retesting is carried out, according to the results of which sick children are identified. The geneticist refers the patient to an endocrinologist, who prescribes treatment and monitors the child in the future.

Galactosemia is one of the hereditary disorders of carbohydrate metabolism. The pathogenesis of the disease is based on a defect in one of the metabolic enzymes - galactose, which is formed in the intestine during the hydrolysis of lactose disaccharide. The first step in the transformation of galactose in the cells of the body is its phosphorylation, which is carried out with the help of the enzyme galactokinase. The product of this reaction, galactose-1-phosphate, is metabolized by galactose-1-phosphate uridyltransferase to uridyldiphosphogalactose. Further transformation of the latter occurs with the help of uridyldiphosphogalactose-4-epimerase. The result of insufficiency of any of the three enzymes - galactokinase, phosphate uridyltransferase or uridyldiphosphogalactose-4-epimerase - an increase in the concentration of galactose in the blood - galactosemia. As a rule, galactosemia means defects in phosphate uridyltransferase, the most severe of which is the classic form of galactosemia. The frequency of the classical form, according to the literature, is 1:50,000-1:60,000 newborns.

There are two forms of galactosemia. Classical galactosemia due to galactokinase deficiency is inherited in an autosomal recessive manner. The gene is localized at the 9p13 locus. The onset of the disease is acute; in the neonatal period, vomiting, diarrhea, jaundice, hepatomegaly, cataracts, malnutrition, psychomotor retardation, and renal tubular dysfunction appear.

Galactosemia due to systemic deficiency of uridyldiphosphogalactose-4-epimerase is also inherited in an autosomal recessive manner. The gene is localized at the 1p36-p35 locus. The onset of the disease is in the neonatal period. Symptoms of the disease are the same, except for cataracts (absent), but there is neurosensory deafness. For these two forms, an asymptomatic benign variant of galactosemia (Duarte's variant) is described, the population frequency of which is higher than the frequency of the classical form.

If treatment is started early, the child will not show clinical symptoms of galactosemia and will grow up healthy. The goal of treatment is to eliminate food products containing galactose, especially breast milk and other milk mixtures. They can be replaced with special mixtures prepared on the basis of soy. Early appointment of treatment, optimally - before the 10th day of life, allows you to avoid severe crises, which often lead to death. Prenatal DNA diagnostics in subsequent pregnancies is possible at the medical genetic consultation.

Cystic fibrosis is one of the most common hereditary diseases, usually severe course and poor prognosis for life. The frequency of cystic fibrosis varies among representatives of the European race from 1:600 ​​to 1:12,000 newborns. The cystic fibrosis gene CFTR is mapped on the long arm of chromosome 7. The number of currently identified mutations in this gene exceeds 2000, of which delF508 is the most common, found in 54% of patients with cystic fibrosis in Russia.

The gene is responsible for the synthesis of a protein that serves as a channel for chloride ions in cells. Due to the dysfunction of this channel, mucus and other secrets in the lungs, pancreas and other organs become very thick and viscous. This leads to the development of chronic infection, damage lung tissue, a violation of the digestion of food, since pancreatic enzymes cannot enter the intestines. The disease usually begins in early age. There are three main forms of cystic fibrosis: pulmonary, intestinal and mixed. The most common of these is a mixed form. It occurs in approximately 80% of patients with cystic fibrosis. The pulmonary form is manifested by a chronic obstructive bronchopulmonary process. A chronic inflammatory process develops, leading to the destruction of lung tissue. The blood of patients is poorly saturated with oxygen, because of which the heart, liver and other organs suffer, children lag behind their peers in height and body weight. Treatment of patients with pulmonary form cystic fibrosis requires the use of powerful antibiotics in large doses. In the intestinal form of cystic fibrosis, the process of digestion of food is disrupted, since pancreatic enzymes that break down proteins and fats do not enter the intestine due to blockage of the ducts of the gland. Basic treatment intestinal form is to take pancreatic enzymes. In the mixed form of cystic fibrosis, intestinal manifestations exacerbate lung damage. Treatment of the mixed form is the most difficult. Patients with cystic fibrosis who do not receive the necessary treatment have a short life expectancy. If cystic fibrosis is detected in a newborn and is treated already from the 2nd month of life, the clinical manifestations of the disease are much easier and the child develops almost normally physically and mentally. It increases life expectancy, which at present, thanks to adequate treatment, is more than 35 years in developed countries.

The screening program uses as a primary test the determination of the content of immunoreactive trypsin in samples of dried blood spots. If the first and second laboratory tests were positive, then, unlike other screened hereditary diseases, this does not mean that the child has cystic fibrosis, although the likelihood of such a diagnosis is high. To confirm the diagnosis, an infant aged 3-4 weeks is given a lot test - measuring the concentration of chlorine in sweat fluid. If the lot test is negative, the child is considered healthy, although he will still be observed for some time. If the lot test is positive, the diagnosis of cystic fibrosis is considered established even before any clinical manifestations of the disease appear.

Adrenogenital syndrome is a group of diseases based on a defect in one of the enzymes or transport proteins involved in the biosynthesis of steroid hormones of the adrenal glands. Most cases of the disease (about 90-95%) are associated with 21-hydroxylase deficiency, 5-10% - with 11-p-hydroxylase deficiency.

The 21-hydroxylase gene (CYP21B) is mapped at the 6p21.3 locus together with the CYP21A pseudogene. A high degree of homology and a tandem arrangement of two genes can lead to their recombination and disruption of the functions of the active gene. Dozens of mutations leading to 21-hydroxylase deficiency have been identified. Point mutations make up about 80%, deletions account for about 20% of the changes. The most frequent point mutations are 12splice, then I172N, etc. The frequency of 21-hydroxylase deficiency is quite high and amounts to 1:8000-1:15,000 newborns. Late diagnosis, untimely and incorrect treatment can lead to serious consequences: the death of a child from salt-losing crises, errors in the choice of gender with severe virilization of the external genitalia in girls, growth disorders, puberty, and infertility. The introduction of neonatal screening makes it possible to detect the disease in a timely manner and avoid diagnostic errors.

There are three clinical phenotypes of adrenogenital syndrome:
salt-losing form - from birth "doubtful" genitals; in the neonatal period - severe salt loss, manifested in the form of adrenal crises (vomiting, dehydration, convulsions, cardiac arrest);
simple virilizing form - from birth "doubtful" genitals in girls, normal in boys, postnatally in both sexes, premature appearance of secondary sexual characteristics, short stature;
attenuated (non-classical) form - onset in puberty and only in girls (poor development of the mammary glands, male-type hair growth, amenorrhea).

These three forms account for approximately 90% of all cases. congenital hyperplasia adrenal cortex, of which 60-65% fall to the share of the salt-losing form. As a result of 21-hydroxylase deficiency, the conversion of cholesterol into cortisol and aldosterone, which is controlled by this enzyme, is impaired. At the same time, there is an accumulation of precursors of cortisol and aldosterone, which normally turn into male sex hormones - androgens. Since a lot of cortisol and aldosterone precursors accumulate in adrenogenital syndrome, much more than normal androgens are formed, which is the main reason for the development of the clinical picture of adrenogenital syndrome. The disease is inherited in an autosomal recessive manner. The screening program uses 17-hydroxyprogesterone concentrations in dried blood spot samples as the primary test. In cases with its high content, retesting is carried out and, thus, sick children are identified. Treatment should be prescribed as soon as possible, then the clinical symptoms of adrenogenital syndrome in the child will not appear and he will grow up healthy, no different from his peers. In medical genetic consultations, prenatal DNA diagnostics can be performed in subsequent pregnancies.

Main stages of neonatal screening
Conventionally, 5 stages of screening of newborns for hereditary diseases can be distinguished.
Stage 1 - taking blood from newborns from the heel in obstetric institutions on the 4th-5th day of life. Standards should be developed and published for all methods of collecting blood samples on filter paper. Ideally, institutions should be provided with video materials. In addition, the system for transporting dried blood samples should be simple and accessible, which will allow for analysis and treatment in a short time, for example, in galactosemia and adrenogenital syndrome within 10 days from birth, before the onset of crises.
2nd stage - rapid primary screening to determine the relevant laboratory parameters. Such analysis is carried out in laboratories with appropriate equipment.
Stage 3 - confirmatory diagnostics with positive results, it must be carried out in the same laboratories as soon as possible. DNA diagnostics and quality control laboratory tests at the 2nd and 3rd stages, they are carried out in federal reference centers.
Stage 4 - treatment of identified patients, which should be carried out by geneticists, neonatologists, pediatricians and endocrinologists. Treatment must be prescribed within the first month of life. Monitoring the effectiveness of treatment is carried out using clinical and laboratory data.
Stage 5 - medical genetic counseling and prenatal DNA diagnostics in families where a sick child has appeared. It is carried out in medical genetic consultations.

All stages must be fully prepared, then you can start the program. For its successful development, many problems must be identified and solved at the stage of program planning. First of all, government support and financial resources are important, since in our country, as in most countries of the world, neonatal screening is a state program.

Directly related to the implementation of the program:
training of maternity hospital staff (collection of blood samples);
laboratory capacity (equipment) and staff training;
the presence of normal values ​​of the studied parameters for the screened population of newborns;
scheme for transporting blood samples;
coordination of work of laboratories;
creating conditions for collecting data and alerting doctors;
creation of a computerized system for storing information about blood samples, conclusions, alerts to parents, identified patients, treatment and its results;
laboratory quality control program;
availability medical care.

An effective way to maintain the quality of all sections of the program is the preparation of practical recommendations with a detailed description of the procedures for each stage of the program. In addition, it is necessary to prepare a general manual (guide) that will summarize practical experience in solving emerging problems. One of the important conditions for the successful implementation of the neonatal screening program is the preparation of the population, the so-called educational block of the program. People need to know what newborn screening is, how it is done, and how it benefits every person in the population.

Three institutions are involved in screening newborns, at least in our country: maternity hospitals (blood sampling from newborns), medical genetic consultations (conducting the 2nd and 3rd stages, treatment of certain diseases and laboratory control of the treatment of all screened diseases , medical genetic counseling of the family), reference centers (laboratory quality control, DNA diagnostics). There is an international network of screening programs.

Laboratory research
GENERAL PRINCIPLES
The biological material used for neonatal screening is blood dried on filter paper.

Obtaining biological material
Blood sampling from each newborn is performed strictly on the 4th-5th day of life (not earlier than 72 hours after birth) in the medical institution where the child is at that moment. By the time of blood sampling, the child must receive at least a day good nutrition. In premature babies, blood is taken on the 7th and 14th day of life. In children who have undergone blood transfusion or hemodialysis, blood sampling is carried out again one month after the last procedure.

Blood sampling is carried out only on special forms of filter paper, currently - Whatman 903. The supply of forms to all medical institutions is carried out by the medical genetic laboratory of the region. The use of any other paper or forms for this purpose is unacceptable, since laboratory measurements, their evaluation and interpretation are carried out using calibration samples and control materials made on the same type of paper. Before taking a blood sample, the heel of the newborn must be washed, wiped with a sterile cloth moistened with 70% ethanol solution, and blotted with a dry sterile cloth. Use of others instead of ethanol disinfectant solutions undesirable, as some of them may influence the measurement result. Blood is taken using a disposable scarifier. The first drop after piercing is removed with a dry sterile cotton swab to avoid hemolysis. Each of the circles indicated on the form is soaked through with one large drop of blood, without touching the child's heel with the form. Blood stains must be at least the size indicated on the form, the appearance of the spots is the same on both sides of the form. This amount of blood is sufficient for screening studies. In case of incomplete filling of the circles with blood, it is necessary to repeat the piercing. Forms with blood are dried for 2-3 hours at room temperature, avoiding direct contact with sun rays. In older children, blood should be taken in the usual way - from a finger.

The following information is clearly and legibly written on the form with blood: the surname, name, patronymic of the mother, if blood is taken in a maternity institution, or of the child, if blood is taken in another medical institution; date of birth of the child, date of blood sampling, detailed registration address and date of departure of the child, telephone number, code of the medical institution and the name of the person who took the blood. Further, related information is recorded: the child's body weight, gestational age, prematurity, the child's blood transfusion, hemodialysis, mother and / or child intake medicines, in particular dexamethasone, hyperbilirubinemia more than 30 mg / dl, etc.

The form is a document that fills in and is responsible for the correctness of blood sampling and the accuracy of the information indicated on the form. Forms with blood are dried at room temperature, packed in a clean paper envelope and delivered to the regional medical genetic laboratory at least once every 3 days. Blood samples taken with abnormalities are assessed as unsuitable for analysis. In this case, it is necessary to perform a second blood sampling.

General principles of the analysis procedure and quality control of laboratory studies
In the medical genetic laboratory that performs neonatal screening in the region, the quality of the obtained biological material is assessed. Forms with blood are sorted and registered in a computer database. Five 3 mm discs are knocked out of each blood sample and then placed in five separate microplates. In each of the microplates, one analyte is measured, which is a biochemical marker of the disease. For phenylketonuria, the marker is the concentration of phenylalanine in the blood, for congenital hypothyroidism - the level of thyroid-stimulating hormone, for cystic fibrosis - immunoreactive trypsinogen, for galactosemia - total galactose, for adrenogenital syndrome - 17-hydroxyprogesterone.

Calibration samples contained in the reagent kit, control materials with a known concentration of the analyte, and test blood samples from newborns are placed in a 96-well microplate. Next, carry out standard procedure analysis in accordance with the kit instructions. The measurement results of each plate are presented in printed form, containing the fluorescence values ​​of the calibrators, the calibration curve, the fluorescence values ​​and analyte concentration in the control materials, the fluorescence and analyte concentration values ​​in the blood samples under study.

Evaluation of measured concentrations of analytes in control materials allows in-house quality control. In a set-up series of at least 20 measurements, each laboratory determines its own mean values ​​and tolerances. The results of measurements of all tablets are entered into the control card. If the values ​​of the control materials meet the requirements set forth in the order of the Ministry of Health of the Russian Federation No. 45 dated February 7, 2000 “On the system of measures to improve the quality of clinical laboratory research in healthcare institutions of the Russian Federation”, the tablet measurement result is assessed as acceptable. Otherwise, the tablet is remade.

Neonatal screening laboratories also participate in the Federal system of external quality assessment, which serves as an external independent control, which is necessary to assess the correctness of ongoing studies and identify systemic errors. Along with this, a number of RF laboratories of this profile are participants in international quality control, in particular CDC.

NEONATAL SCREENING FOR Phenylketonuria
Screening for phenylketonuria is the standard of neonatal screening programs, since for more than 40 years of its implementation, a huge amount of material has been accumulated on the etiology of the disease, laboratory methods of diagnosis and treatment. Currently, there is a wide range of methods for measuring the concentration of fibroadenoma, ranging from the microbiological inhibitor test used so far to tandem mass spectrometry. In the Russian Federation, the determination of the level of fibroadenoma in dry blood spots is carried out by a microplate fluorimetric method. The principle of measuring the concentration of fibroadenoma in a dry blood spot is based on the formation of a fluorescent complex of fibroadenoma with ninhydrin, the fluorescence intensity of which increases when interacting with the dipeptide L-leucyl-b-alanine. Fluorescence is measured using a multifunctional analyzer at a wavelength of 485 nm. Fluorescence intensity is directly proportional to the amount of fibroadenoma in a blood sample. The software compares the fluorescence intensity of the analyzed blood samples with the fluorescence of the calibration samples. The correctness of the analysis is assessed by the values ​​of fibroadenoma in control samples.

Interpretation of results
Fundamental is the threshold value of fibroadenoma concentration, produced by the laboratory, taking into account the set of reagents recommended by the manufacturer, the population values ​​of the analyte level for newborns in this region, as well as information from similar laboratories in the Russian Federation and abroad. To select this indicator, it is important to estimate the number of retests, which depends on the cut-off value. For newborns and children of the first month of life, the level of fibroadenoma equal to 2 mg / dl (120 μmol / l) is most often taken as the threshold, for children older than one month - 3 mg / dl (150 μmol / l). Blood samples in which the result of the first fibroadenoma measurement was abnormally high are analyzed additionally in a parallel analysis using the same blood sample. All children who have parallel dimension the level of fibroadenoma turned out to be higher than the cut-off value, they are subject to re-examination.

Obtaining a second blood sample from a child (retest) is carried out at the place of residence or in the medical institution where he is located. To do this, depending on the degree of increase in the concentration of the amino acid, using the information on the form with blood, contact is established with the family. If the excess level of fibroadenoma is insignificant - up to 3 mg / dL (181.5 μmol / L), the family is notified by letter about the need for a second study. With a significant increase in fibroadenoma - more than 3 mg / dl - there is a need for emergency contact with the family. Local control over the provision of retests is carried out by the chief pediatrician of the health department of the district, city, with whom the laboratory is in constant contact by phone or via e-mail.

In most cases, especially in children with a small primary increase, the level of fibroadenoma on retest analysis is normal. The initial increase in the indicator could be associated with the immaturity of the liver enzyme systems, the characteristics of the course of labor, prematurity, and severe general condition child, etc.

Children with a repeated increase in the concentration of fibroadenoma in the retest from 3 to 8 mg / dl (150-484 μmol / l) are diagnosed with hyperphenylalaninemia. They need regular laboratory monitoring of the level of fibroadenoma and observation by a geneticist who decides whether the treatment is appropriate or not. If a fibroadenoma level equal to or more than 8 mg/dl (484 µmol/l) is detected in the retest, the diagnosis of phenylketonuria is considered confirmed, since this indicator serves as a reliable laboratory criterion for the disease. Parents with a child are invited to a medical genetic consultation. The geneticist promptly prescribes the appropriate treatment for the child with the restriction of fibroadenoma and teaches parents how to calculate the diet. According to modern standards, the diagnosis of phenylketonuria should be made and treatment should not be started. later than a month child's life. Subsequent treatment, carried out for many years, is carried out under constant biochemical control of the level of fibroadenoma in the blood, also performed by the medical genetic laboratory. The optimal concentration of the amino acid in the blood during treatment is considered to be between 1 and 6 mg/dL (60.5-363 µmol/L).

NEONATAL SCREENING FOR CONGENITAL HYPOTHYROISIS
The etiology of congenital hypothyroidism is different, but all its forms are characterized by insufficiency of thyroid hormones. Due to the low specificity and blurring of clinical symptoms in newborns, early diagnosis of congenital hypothyroidism is possible only on the basis of a study of the level of thyroid hormones. Screening is based on determining the level of thyroid-stimulating hormone, which increases in the primary forms of the disease. Measurement of the level of thyroid-stimulating hormone in dry blood spots is carried out by the method of lanthanide immunofluorescent analysis with time resolution, using a "sandwich" of highly specific monoclonal antibodies against two different sites on the thyroid-stimulating hormone molecule. The level of fluorescence is stable, its intensity is directly proportional to the amount of thyroid-stimulating hormone in the sample.

Interpretation of results
The interpretation of the values ​​of thyroid-stimulating hormone obtained during screening is carried out taking into account the set of reagents recommended by the manufacturer and population data on the level of thyroid-stimulating hormone for newborns in the region. For analyzes taken on the 4th-7th day of a child's life, the cut-off is 14 μIU / ml, for children over the age of 14 days - 5 μIU / ml. 80 µIU/mL is used as a threshold value to suspect hypothyroidism with a high degree of probability. All children with thyroid-stimulating hormone levels above this value are subject to re-examination, i.e., to be called for a retest on an emergency basis. The re-collected blood of such children must be delivered to the laboratory within 48 hours after collection. Children with a re-detected increase in the level of thyroid-stimulating hormone are referred to an endocrinologist for verification of the diagnosis (congenital or transient hypothyroidism) and treatment.

NEONATAL SCREENING FOR GALACTOSEMIA
Currently, various algorithms (together or separately) are used as screening schemes for galactosemia: measurement of the concentration of galactose and galactose-1-phosphate, analysis of the enzymatic activity of galactose-1-phosphate uridyltransferase.

The use of blood galactose concentration as a diagnostic criterion allows simultaneously with a defect in galactose-1-phosphate uridyltransferase to detect deficiencies of galactokinase and uridyldiphosphogalactose-4-epimerase, since the concentrations of these analytes are increased in all three cases. However, with the introduced restriction in the diet, this indicator is not informative.

The advantage of the analysis of the enzymatic activity of galactose-1-phosphate uridyltransferase is its independence from the nature of nutrition and food restrictions, if any, introduced before the examination. However, in the case of a previous blood transfusion, a false negative result may be obtained. On the other hand, the conditions for obtaining, transporting and storing the material (temperature, humidity) can lead to a decrease in the activity of the thermolabile enzyme, i.e., to a false positive result.

Some foreign neonatal screening programs use molecular genetic methods to search for the most frequent mutations in the galactose-1-phosphate uridyltransferase gene. This search is performed in parallel with the study of biochemical parameters or as a subsequent step in the study of blood from the same form. The use of DNA analysis makes it possible to optimize screening - to reduce the number of false positive results, to differentiate the classical form, etc. However, the ability to identify a limited number of mutations does not make it possible to cover all variants of the disease. The level of galactose is elevated in all forms of galactosemia. That is why this criterion is used as a primary biochemical indicator.

The measurement of total galactose in dry blood spots is carried out by a microplate fluorimetric method. The galactose oxidase method used makes it possible to quantify the concentration of total galactose, i.e. the sum of the concentrations of free galactose and galactose-1-phosphate. The correctness of the analyte measurement is assessed by carrying out intralaboratory quality control to determine its concentration in control materials.

Interpretation of results
The interpretation of the values ​​of total galactose obtained during screening is carried out taking into account the cut-off, which is developed, focusing on the set of reagents recommended by the manufacturer, the population data of the region, and the available experience. As a threshold concentration of total galactose for newborns in most laboratories of the Russian Federation, the value of 7 mg / dl (385 μmol / l), recommended by the manufacturer of the test system, is accepted. Children with analyte concentrations greater than 7 mg/dl in blood samples should be retested.

The urgency of obtaining a second blood sample from a child (retest) depends on the degree of increase in total galactose. Since the classic form of galactosemia is characterized by an acute, severe manifestation in the neonatal period, life-threatening, with a total galactose value exceeding 15 mg / dl (825 μmol / l), it is urgent to contact the family and the main pediatrician of the territory to obtain information about the child's condition and receiving a retest. If an elevated level of this indicator is confirmed in the retest and based on the state of health of the child, the neonatologist or pediatrician may decide to urgently transfer the child to a galactose-free diet without waiting for the results of laboratory verification of the diagnosis.

An elevated concentration of galactose in the blood is a necessary but not sufficient criterion for diagnosing galactosemia. A slight increase in analyte concentration is characteristic of the Duarte form. In addition, the peculiarities of the course of childbirth, the severe general condition of the child, the insufficiency of liver function, and chromosomal diseases lead to an increase in the level of galactose in the blood.

Clarification of the form of galactosemia requires mandatory additional examination- studies of the enzymatic activity of galactose-1-phosphate uridyltransferase and analysis of mutations in the gene galactose-1-phosphate uridyltransferase or its sequencing. Since DNA analysis allows the study of a limited number of mutations, the detection of a deficiency in the enzymatic activity of galactose-1-phosphate uridyltransferase is an important diagnostic criterion. If the diagnosis is confirmed, the child is urgently prescribed a galactose-free diet and medical genetic counseling of the family is carried out. Further treatment of the child is carried out under the control of the determination of galactose in the blood.

NEOTHATAL SCREENING FOR CYS FICIDOSIS
There are a number of schemes for neonatal screening for cystic fibrosis, the first stage of which is the determination of the level of immunoreactive trypsinogen. Trypsinogen is one of the main secretion products of the pancreas, the only one of the enzymes that is produced only by the pancreas, so it is a specific marker of pancreatic function. In cystic fibrosis, an increase in the level of immunoreactive trypsinogen in the blood is observed in the first 2 months of a child's life. Further, the level of immunoreactive trypsinogen decreases and reaches the average population values.

The results obtained by measuring the level of immunoreactive trypsinogen are further complemented by a sweat test and/or DNA analysis in various combinations:
IRT -> sweat test -> DNA analysis;
IRT -> DNA analysis -> sweat test;
IRT 1 -> IRT 2 -> sweat test -> DNA analysis.

In Russia, the latter scheme is generally accepted. Measurement of the level of immunoreactive trypsinogen in dry blood spots is carried out by the method of lanthanide immunofluorescence analysis with time resolution, using a "sandwich" of highly specific monoclonal antibodies against two different sites on the immunoreactive trypsinogen molecule. Fluorescence is stable, its intensity is directly proportional to the amount of immunoreactive trypsinogen in the sample.

Interpretation of results
The interpretation of the values ​​of immunoreactive trypsinogen obtained during screening is carried out taking into account the cut-off, a set of reagents recommended by the manufacturer and the Russian Center for Cystic Fibrosis. For children under the age of 21 days, values ​​of immunoreactive trypsinogen up to 70 ng / ml are considered normal. For older children, the cut-off is 40 ng/mL.

Immunoreactive trypsinogen is not a specific marker of the disease; it is impossible to make a diagnosis based on its primary value. That is why all children with elevated levels of immunoreactive trypsinogen need to be re-examined. For re-examination for cystic fibrosis (obtaining a retest), there is a strictly limited period: from 21 days to 2 months of life. Blood taken at a later age is unsuitable for research due to the non-informative nature of the test. The diagnosis must be removed or confirmed by other methods. An increase in the level of immunoreactive trypsinogen may be due to the peculiarities of the course of labor - a long anhydrous period, rapid labor, as well as the peculiarities of the course of the postpartum period - neonatal stress, respiratory distress syndrome, hypoglycemia, congenital infections, intestinal atresia, severe congenital and chromosomal diseases, etc.

Sweat test and DNA analysis
Children with a detected increase in immunoreactive trypsinogen in the retest, as well as those who have not been re-examined for immunoreactive trypsinogen by age, need the second stage of screening - a sweat test. Sweat test - measurement of the concentration of chlorine in sweat fluid - the main pathognomonic diagnostic criterion for cystic fibrosis. The classic, but time-consuming and labor-intensive method of performing a sweat test is the determination of the concentration of chlorine in sweat by titrating it according to the method of Gibson and Cook. Currently, the study is carried out using an apparatus that measures the electrical conductivity of sweat, equivalent to the concentration of chlorine. Sweat is collected on the child's forearm with preliminary pilocarpine electrophoresis at the collection site. The normal range of chlorine concentrations recommended by the manufacturer of the reagents is 0-60 mmol/l of sweat. Values ​​of 61-80 mmol / l are considered doubtful, requiring re-checking, repetition and clinical observation in dynamics. Chlorine concentrations greater than 80 mmol/l are associated with cystic fibrosis.

In parallel with the measurement of immunoreactive trypsinogen, an analysis of frequent mutations, in particular delF508 and others following in frequency, is performed, which is an important diagnostic criterion for this disease and optimize neonatal screening. However, since the number of known mutations in the gene is large, the study of frequent mutations does not always confirm or refute the diagnosis. That is why the diagnosis should always be confirmed by a sweat test. Children with a confirmed diagnosis are referred for treatment and dispensary observation in regional center. A geneticist provides medical genetic counseling to the family.

NEONATAL SCREENING FOR ADRENOGENITAL SYNDROME
The first step in neonatal screening for adrenogenital syndrome is to determine the level of 17-hydroxyprogesterone, which is a precursor to cortisol. The level of 17-hydroxyprogesterone is elevated in both forms of adrenogenital syndrome caused by a deficiency of 21-hydroxylase or 11-b-hydroxylase, which makes it possible to identify more than 95% of children with adrenogenital syndrome. In other forms of adrenogenital syndrome, the level of 17-hydroxyprogesterone does not change, but the frequency of these forms is low. Determination of the level of 17-hydroxyprogesterone in dry blood spots is carried out by the method of lanthanide immunofluorescent analysis with time resolution. The test is based on the competition of europium-labeled 17-hydroxyprogesterone and 17-hydroxyprogesterone in newborn blood for the binding site with monoclonal antibodies specific for 17-hydroxyprogesterone. Fluorescence is stable, its intensity is inversely proportional to the amount of 17-hydroxyprogesterone in the sample.

Interpretation of results
The interpretation of the obtained values ​​of 17-hydroxyprogesterone is carried out taking into account the set of reagents recommended by the manufacturer and the Research Institute of Pediatric Endocrinology SE ENTS RAMS. For full-term babies with a gestation period of more than 37 weeks and a body weight of more than 2000 g, cut-off 17-hydroxyprogesterone in the blood is 30 nmol / l. As a threshold value to suspect adrenogenital syndrome with a high degree of probability, a value of 90 nmol / l is used.

For premature babies with a gestational age of 33-36 weeks and a body weight of less than 2000 g, the threshold level of 17-hydroxyprogesterone is 60 nmol / l. In children with deep prematurity (gestational age - 23-32 weeks), the result is considered positive if the level of 17-hydroxyprogesterone is more than 150 nmol / l.

In addition to prematurity, false positive results can be obtained in children with a severe general condition, against the background of intravenous transfusion, with high bilirubinemia (more than 30 mg / dl). In children receiving dexamethasone (or if the mother takes the drug), a false negative result may be obtained. Children with a repeated increase in the level of 17-hydroxyprogesterone in the retest are referred to a pediatric endocrinologist to verify the diagnosis and prescribe treatment. All children with a diagnosis of adrenogenital syndrome, their parents and family members need a molecular genetic study and medical genetic counseling.

In conclusion, it should be emphasized that any screening program using modern algorithms and laboratory methods does not detect 100% of patients with this disease, which is objectively due to various forms diseases, insufficient sensitivity and specificity of the methods used, organizational deficiencies, human factor, etc. That is why, with any clinical suspicion of a disease, the patient must be re-examined in full.

For citation: Kusova Z.A., Kashirskaya N.Yu., Kapranov N.I. Peculiarities of mass screening of newborns for cystic fibrosis // RMJ. 2010. No. 5. S. 265

The first attempts to conduct neonatal screening (NS) for cystic fibrosis (CF) in Europe were made in the early 1970s and were limited to determining the content of albumin in meconium. And only the increase in the level of immunoreactive trypsin (IRT) in the blood plasma of newborns with CF, discovered in 1979, was the impetus for the start of mass screening of newborns for this disease. Further improvement of the NS program became possible after the cloning of the CFTR gene in 1989 and the subsequent identification of specific CFTR mutations in the general population, which made it possible to include DNA analysis in screening protocols. Every year, more than 1.6 million newborns were examined in Europe under the NS program and more than 400 sick children were identified. According to 2008 data, the number of children screened exceeded 3 million per year due to the introduction of HC in CF in the UK and Russia. The NS program is justified both from a medical and economic point of view. Early diagnosis CF makes it possible to start adequate therapy in a timely manner, which leads to a significant improvement in the quality and life expectancy of patients. In addition, conducting NS and establishing the CFTR genotype of newborns with CF suggests the possibility of earlier genetic counseling, which may affect the reproductive behavior of spouses and their relatives.

At present, there are about 26 variants of NS programs in Europe, including from 2 to 4 successive stages of examination (Table 1). The first step in all protocols is to determine the level of IRT in the dried blood spot of a newborn in the first week of life: a very sensitive (85-90%), but not specific sign. According to the European Consensus, hypertrypsinemia in the neonatal period occurs with perinatal stress, conjugative jaundice of newborns, with trisomies 13 and 18, in children with congenital infections, renal failure and atresia of the small intestine, as well as in the case of nephrogenic diabetes insipidus. The population distribution of IRT blood concentrations during the neonatal period is slightly higher in children of North African descent and in African Americans than in children from Northern Europe. Therefore, a second stage of examination is necessary.
Using the RTI/DNA approach in a multiethnic society does not allow identification of patients with mutations specific to some ethnic groups. In the European Mutation Study in North African and Turkish CF patients, only 50% of mutations were detected using standard mutation panels. This presents a problem for countries and/or large cities with numerous ethnic groups. Some current IRT-DNA-based NA programs attempt to compensate for this by keeping a second IRT sample in children who did not have a CFTR mutation, but the IRT level in the first sample was very high.
The use of pancreatitis associated protein (PAP) as a Level 2 test, or in combination with RTI determination as a Level 1 test, is being studied. This approach will avoid the problems associated with the analysis of CFTR mutations or the need for repeated blood sampling. A combined RAD + RAP assessment kit has been developed and pilot studies are planned in the Netherlands, Germany and France (Jeannette Dan-kert-Roelse, Olaf Sommerburg and Jacques Sarles, personal communication) .
All of the above programs can and should be combined and carried out in relatives identified by screening, both in families with CF patients and in the population as a whole (cascade screening). Since siblings (brothers and sisters of patients) have a 50% chance of being carriers (and aunts and uncles have a 25% chance), this cascade screening method can be effective and associated with minimal costs. NS also has a number of negative aspects. The moment parents first hear about a positive screening result for their child can be a critical time, both medically and psychologically. The primary purpose of providing information during this period is to ensure timely provision of medical attention and a confirmatory sweat test, unless the child has a homozygous or mixed heterozygous CF mutation that is indicative of a definite diagnosis. However, during this period of “maximum uncertainty,” it is understandable that parental anxiety can be significant. Minimizing the interval between the initial discussion of the CF screening result and diagnostic confirmation is beneficial for psychological state as well as the initiation of medical care and the development of mutual trust between the patient's family and health care workers.
As stated in the European Consensus, the goal of CF NA is to identify the largest possible proportion of CF patients with a minimum number of false positives at an affordable cost. This can be achieved by using different screening protocols. Since the priorities for newborn screening in many countries and regions differ in terms of funding, ease of blood sample collection, ease of access to clinical services, and prevalence of CFTR mutation, it is not possible to achieve full harmonization of protocols. The choice of strategy depends on population genetics, cost, focus on specific goals: maximum sensitivity, minimum or no need for repeat blood sampling, frequency of unnecessary detection of carriers, and reduction in sweat sampling. Central to the success of newborn screening for CF is effective communication between healthcare professionals and parents. The communication standard should cover pre-screening information for families, as well as information for parents of children with a positive screening of newborns, newborns with CF, and carriers.
Since 2006, in a number of regions, and since January 1, 2007, in all regions of the Russian Federation, mass screening of newborns for cystic fibrosis has been included in the list of hereditary diseases subject to mandatory NS along with phenylketonuria, galactosemia, hypothyroidism and adrenogenital syndrome within the national priority project " Health". The screening protocol includes 4 stages: IRT, IRT2, sweat test and DNA diagnostics, and only the first three are mandatory (Table 2).
Genetic examination in the Russian Federation is carried out only in a number of regions. Its availability is limited by the high cost of analysis (for example, 3500 rubles for 26 mutations of the CFTR gene, which is 70-75% of total number mutant alleles of the CFTR gene found in CF patients in Russia).
The sweat test is the "gold standard" of the CF screening protocol. The UK Association of Clinical Biochemists recommends that a minimum of 50 sweat samples per year be performed at each center responsible for diagnosing CF. Currently, most European centers continue to measure the concentration of chlorides in sweat (the direct classical Gibson-Cook biochemical method). Two systems for the analysis of sweat conductivity (indirect determination of chlorides) are registered and successfully used in the Russian Federation. The Macroduct sweat collection and analysis system in combination with the Sweat-Chek sweat analyzer from Wescor (USA) allows you to conduct a sweat test outside the laboratory, the sweat collection time is 30 minutes, it is successfully used in children from the first months of life. Especially for the examination of newborns, Vescor developed the Nanoduct apparatus, which combines a system for stimulating sweating by electrophoresis of 0.1% pilocarpine and a sweat conductivity analyzer. Due to the minimum amount of sweat fluid required for the test (only 3-6 µl), this device is indispensable when examining newborns as part of mass screening. It is important to remember that the conductivity of sweat is determined by the totality of all ions present in the sweat fluid (potassium, sodium, chlorine, bicarbonate, ammonium, etc.), and the result obtained exceeds the true concentration of chlorides by about 15-20 mmol / l. Thus, results above 80 mmol / l are considered positive, and indicators of 60-80 mmol / l are borderline (Table 3).
An important achievement in practical healthcare is the centralized purchase of devices for the Nanoduct sweat analyzer of the Ministry of Health of the SR RF for all subjects of the Russian Federation. Specialists from the regions were trained to work on the devices at the Russian Center for Cystic Fibrosis.
According to the Ministry of Health and Social Development of the Russian Federation, from January 1, 2007 to December 31, 2009, 4,160,021 newborns were examined for CF in the Russian Federation. According to data obtained from all regions of the Russian Federation, 416 cases of CF were identified. The preliminary frequency of the disease in Russia is 1:10,000 newborns. It should be noted that not all children with repeated high RTI values ​​undergo sweat tests, because for various reasons, parents refuse this study(up to 25% in different regions). Thus, the true frequency of CF in Russia is much higher than the indicated value. Taking into account the data obtained from different subjects of the Russian Federation, it can be argued that this frequency also varies significantly by region (Table 4).
Newborns with an established diagnosis are regularly observed by specialists of the MV Center: every 2 weeks. up to 3 months child's life, monthly up to six months, every 2 months. from six months to 1 year and then quarterly (Table 5). Of particular importance is the monthly dynamic surveillance for patients without clinical manifestations - weight and height indicators, the results of a coprological study (at least 1 time per month up to 1 year), indicators of pancreatic elastase in the stool (2 times in the first year of life), the growth of microflora in the sowing of a swab from the oropharynx and a clinical blood test (1 time in 3 months). In the event of an exacerbation of the bronchopulmonary process or the absence of the desired control over the symptoms of the disease, a deeper examination may be required (X-ray examination of the lungs or CT scan, lipidogram of feces, biochemical analysis blood, proteinogram, etc.).
Treatment of a child with CF should begin as soon as the diagnosis is made. The volume of therapy depends on the clinical manifestations and the results of laboratory and instrumental methods of examination. In 90% of patients with CF, the first clinical manifestations occur in the first year of life and, as a rule, in the first months. All newborns and children in the first months of life with CF are shown to start early kinesitherapy, regardless of whether they have signs of broncho-pulmonary lesions. In infants, a passive kinesitherapy technique is used, including therapeutic positions, contact breathing, light vibration, stroking, and ball exercises. At this stage, close contact with the child is very important, all activities should be enjoyable for the baby. In children with the slightest symptoms of bronchial obstruction, kinesitherapy is used in combination with mucolytic drugs and bronchodilators.
According to Verhaeghe C. et al. from Belgium, in the lung tissue of fetuses with CF, a significant increase in the level of pro-inflammatory proteins was noted, which indicates an early onset of inflammatory processes that precede the development of infection. That is why, in our opinion, the early prescription of dornase alfa (Pulmozyme, F. Hoffmann-La Roche Ltd.) is justified due to the presence of this drug, along with a good mucolytic effect, of an anti-inflammatory action, characterized by a decrease in inflammation markers in the bronchoalveolar fluid (neutrophil elastase, IL-8).
All newborns with CF who have clinical manifestations of intestinal syndrome or low levels of fecal elastase-1 (activity may vary during the first year of life) are indicated replacement therapy microspherical pancreatic enzymes under the control of the coprogram, the frequency and nature of the stool, monthly weight gain. Mandatory is the appointment of fat-soluble vitamins.
Currently, 42 children with CF are being observed in the Moscow CF Center, identified under the NS program from June 2006 to March 2010 (Table 6).
In 2009, in order to determine the effectiveness of NS on CF, we analyzed CFTR mutations in dried blood spots of 990 newborns with the first positive test for RTI, who were born in 2008 in Moscow and were at risk for CF. During the study, CFTR mutations were found in 47 in-di-species. The number of mutant alleles of the CFTR gene was 53, or 2.7%, and was represented by the following CFTR mutations: F508del - found in 28 cases (68%), CFTRdele 2.3 (21kb) - in 7 (17%) cases , 2184insA - in 2 (5%) cases, 3821delT - 1 (4%) , L138insA - 2 (4%), 2143delT - in 1 (2%) DNA sample.
The study identified a girl aged 1 year 4 months with the CFTRdele2.3(21kb)/CFTRdele2.3 (21kb) genotype, who was not included in the risk group according to NA (RTI I - 236 ng/ml, RTI II - 12 ng/ml). ml) and was not diagnosed in a timely manner. The child's family was invited for a consultation at the CF center. The sweat test result is 112 mmol/l. At the time of examination, the child's weight and height indicators corresponded to the age norm, in the anamnesis - repeated acute respiratory viral infections, fatty stools, hospitalization with suspected acute intestinal obstruction.
Thus, we found that the frequency of mutant alleles of the gene in newborns at risk for CF (with the first positive RTI) is 0.02575 (0.02017 ÷ 0.03241), which is significantly higher than the frequency of these mutations in the Russian population (0.00642 ). It is possible that heterozygous carriage of F508del, CFTRdele2.3 (21kb), 3821delT, L138insA, 2143delT, 2184insA mutations in the CFTR gene, identified during the study, has an effect on the increase in the level of IRT, as a result of functional pancreatic insufficiency in newborns .
The proportion of false-negative results of NS on CF in 2008 was 0.1%, which does not contradict the pan-European data.
Most specialists in the field of CF come to the conclusion that the NS on CF is justified, firstly, from an economic point of view, since it helps to prevent the birth of patients with CF in families where there is already a sick child, and contributes to the appearance of healthy children in these families; secondly, from a medical point of view, since the life expectancy of patients identified through screening is higher than in other groups. In addition, screening reduces the time of sometimes painful diagnosis.
The European MV Association has created working group for neonatal screening, in 2007 it included representatives from Russia. The main task of the group is data analysis different countries and European regions, which, in turn, may contribute to the optimization of screening programs in the future.
The value of screening for CF in Russia as a whole can only be assessed in a few years, provided that the program is regularly funded. In addition, for tangible results comparable to European or American ones, it is necessary for the state to understand the importance of not only the timely detection of CF patients, but also the creation of the necessary conditions for their observation and treatment.