RU2790923C1 - METHOD FOR MORPHOLOGICAL DIAGNOSIS OF A FUNCTIONING DUCTUS ARTERIOSUS IN PREMATURE NEWBORNS WITH BIRTH WEIGHT BELOW 1500 g - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: it can be used for post-mortem morphological diagnosis of a functioning ductus arteriosus in premature new-borns with a birth weight below 1500 g. Prostaglandin E2 expression index in the ductus arteriosus wall is determined by the formula ∑P(i) ⋅ i/100, where i is the intensity of staining in the immunohistochemical reaction of sections of the arterial duct, assessed in 10 fields of view of the microscope in 100 cells of the preparation at x 400 magnification expressed as score from 0 to 3; P(i) is the percentage of cells stained with different intensities. If the expression index of the prostaglandin E2 receptor is equal to or more than 1.5 conventional units, the functioning ductus arteriosus is diagnosed, and if the value is less than 1.5 conventional units, the non-functioning ductus arteriosus is diagnosed.

EFFECT: effective post-mortem morphological diagnostics of a functioning ductus arteriosus in premature new-borns at the gestational age of 22-32 weeks and birth weight less than 1500 g by determining the expression index of the prostaglandin E2 receptor in the ductus arteriosus wall.

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Description

The invention relates to medicine, namely to pathological anatomy, and can be used for post-mortem diagnosis of a functioning ductus arteriosus (FAP) in very premature newborns with a gestational age of 22-32 weeks and a birth weight of less than 1500 g.

The frequency of occurrence of a functioning ductus arteriosus (code according to the International Statistical Classification of Diseases and Related Health Problems, tenth revision (ICD-10) R 29.3) is inversely related to gestational age. In very premature babies, this pathology is observed in 25 - 70% [Modern view of the functioning ductus arteriosus in premature newborns / Zh.A. Zvyagina, E.G. Tsoi, L.N. Igisheva, D.A. Tronina // Mother and child in Kuzbass. - 2018. - T. 75, No. 4. - S. 4-12].

In newborns with a birth weight of less than 1500 g, there is often a violation of the mechanisms for closing the arterial duct. Even if after birth there is a functional closure of the duct, then this category of children has all the prerequisites for its re-opening [Modern ideas about the open ductus arteriosus in newborns / D.S. Kryuchko, A.G. Antonov, A.A. Lenyushkina and others // Pediatrics. Journal them. G.N. Speransky. - 2011. - T. 90, No. 1. - S. 130-136]. Hyperventilation of the lungs, anemia, sepsis, pneumothorax, and others can be conditions that contribute to the re-opening of the duct.

In preterm infants, FAP may be associated with a critical clinical condition and play a decisive role in their thanatogenesis. Hemodynamic disorders in a functioning ductus arteriosus lead to a change in cerebral blood flow and the development of early intraventricular hemorrhages, which are one of the main causes of neonatal mortality in the perinatal period [Modern view of the functioning ductus arteriosus in premature infants / Zh.A. Zvyagina, E.G. Tsoi, L.N. Igisheva, D.A. Tronina // Mother and child in Kuzbass. - 2018. - T. 75, No. 4. - S. 4-12].

Thus, the development of a method for the morphological diagnosis of a functioning ductus arteriosus in very premature newborns will improve the quality of the histological conclusion in the morphological study of the ductus arteriosus and objectively and reliably assess not only its presence, but also its functioning at the molecular level.

A known method for diagnosing patent ductus arteriosus in premature infants 25 - 34 weeks of gestation by the level of BNUP in the blood [Yoo K.H., Hong Y.S., Son C.S., Lee J.W. Utility of rapid B-type natriuretic peptide assay for diagnosis of symptomatic patent ductus arteriosus in preterm infants / B.M. Choi, K.N. Lee, B.L. Eun et al. // Pediatrics. - 2005. - Vol. 115, No. 3. - P. 255-261]. At a BNUP concentration of 1110 pg/ml or more, a functioning ductus arteriosus is diagnosed.

However, this method has the following disadvantages:

1. The method is intended for intravital diagnostics and cannot be used for post-mortem diagnostics.

2. The method does not allow to diagnose a functioning ductus arteriosus at the morphological level.

A known method for the diagnosis of FAP by the level of BNUP in the blood of premature babies less than 37 weeks of gestation [Kim J.S., Shim E.J. B-Type Natriuretic Peptide Assay for the Diagnosis and Prognosis of Patent Ductus Arteriosus in Preterm Infants // Korean Circ J. - 2012. - Vol. 42. – P. 192-196]. At a BNUP concentration level in the blood of 412 pg/ml or more, a functioning ductus arteriosus is diagnosed.

However, this method has the following disadvantages:

1. The method is intended for intravital diagnostics and cannot be used for post-mortem diagnostics.

2. The method does not allow to diagnose a functioning ductus arteriosus at the morphological level.

A known method for diagnosing FAP by echocardiography, according to which the main criteria for the hemodynamic significance of the patent ductus arteriosus include duct diameter >1.5 mm (with body weight <1500 g) or >1.4 mm (with body weight >1500 g), and also shunting of blood from left to right. Additional criteria include signs of overflow of the pulmonary circulation: the ratio of the size of the left atrium to the aortic root (LA / Ao) ≥1.5, diastolic blood flow velocity in the pulmonary artery (LA) ≥0.42 m/s, the ratio of cardiac output of the left ventricle ( LVV) to superior vena cava blood flow (LVO/SVC) >4.0, LVV ≥300 mL/kg/min, left ventricular (LV) end-diastolic size to aortic root size ratio (LVd/Ao)>2.1, and criteria for impoverishment of the systemic circulation (vascular resistance index (Ri) of the anterior cerebral artery > 0.8, retrograde blood flow in the renal and/or mesenteric arteries (pendulum blood flow), retrograde blood flow in the postductal aorta > 50% of antegrade blood flow [Public ductus arteriosus in preterm infants children / A. A. Burov, D. N. Degtyarev, O. V. Ionov et al. // Neonatology: news, opinions, training. - 2016. - V. 14, No. 4. - P. 120-128] .

However, the method has several disadvantages:

1. In extremely premature newborns, the level of hypoxia of the arterial duct wall does not reach the degree at which its remodeling occurs, as a result, re-opening or recanalization of the vessel may occur [Open ductus arteriosus in premature newborns / D.S. Kryuchko, E.N. Baibarina, A.G. Antonov, A.A. Rudakova // Questions of practical pediatrics. - 2010. - V. 5, No. 2. - S. 57-65]. Therefore, echocardiography data in this category of children in the neonatal period have diagnostic value only at the time of the study.

2. The method is intended for intravital diagnostics and cannot be used for post-mortem diagnostics.

3. Does not allow to diagnose a functioning ductus arteriosus at the morphological level.

The closest technical solution is a method for determining the patency of the arterial duct using a probe during a pathoanatomical autopsy [Avtandilov G.G. Fundamentals of pathoanatomical practice: a guide. - M.: RMAPO, 1994. - 512 p.]. To do this, putting the heart on the table with the base to the doctor, the pulmonary artery is opened along the anterior wall from the valve opening and the place where the ductus arteriosus originates is examined. Further, to determine the patency of the vessel, a buttoned or grooved probe is inserted into the duct. If the duct is passable, then the probe penetrates into the lumen of the aortic arch.

The method has a number of disadvantages:

1. According to our studies, the median diameter of the functioning ductus arteriosus in very preterm infants who died in the neonatal period with birth weight less than 1500 g is 1.2 [0.35; 1.8] mm, while the standard sectional instruments used to diagnose the patency of the anterior duct have a diameter much larger than this value (the diameter of the bellied probe is 2 mm, the grooved probe is 4 mm). Due to the discrepancy between the diameters, these probes cannot pass the open ductus arteriosus, which is why a false opinion is formed about the non-functioning of this vessel at the time of death. There is an underestimation of the role of the functioning ductus arteriosus in thanatogenesis, which can distort and reduce the quality of the pathoanatomical conclusion.

2. Due to the small size of the macropreparation of the heart of very premature newborns and the narrowness of the arterial duct, there is a risk of its perforation when passing through standard sectional probes, while a false opinion is formed about the functioning of the arterial duct at the time of death, an overestimation of the role of the functioning arterial duct in thanatogenesis, which can distort and reduce the quality of the pathoanatomical conclusion.

technical solution The proposed method is post-mortem morphological diagnosis of a functioning ductus arteriosus in very premature newborns with a gestational age of 22-32 weeks and with a birth weight of less than 1500 g.

The technical result is achieved by determining the expression index of the prostaglandin E2 receptor in the wall of the ductus arteriosus in dead premature newborns with a gestational age of 22-32 weeks and a birth weight of less than 1500 g, with a value equal to or more than 1.5 conventional units, a functioning ductus arteriosus is diagnosed, and with a value of less than 1.5 conventional units - a non-functioning ductus arteriosus.

A morphological study of 42 arterial ducts obtained during autopsy of newborns who died in the neonatal period, born with a body weight of less than 1500 g at a gestational age of 22-32 weeks, was carried out. The physical development of newborns corresponded to the gestational age. As the main cause of death, they were diagnosed with bilateral non-traumatic intraventricular hemorrhages of II-III degree and congenital interstitial pneumonia. Children with myocarditis, congenital malformations of the heart and blood vessels were excluded from the survey. During the pathoanatomical autopsy, all children underwent isolation of the arterial duct and determination of its patency. For these purposes, polyurethane central venous and umbilical catheters were used with sizes from 1 to 5 Fr with a diameter of 0.35 - 1.8 mm (1 Fr - diameter 0.35 mm; 2.5 Fr - 0.8 mm; 3.5 Fr - 1.2 mm; 4 Fr -1.5 mm; 5 Fr - 1.8 mm, Vygon, Germany). In 28 children, the ductus arteriosus was passable for a catheter (of which 2 children - for a 1 Fr catheter, 7 children - 2 Fr, 5 - 3 Fr, 9 - 4 Fr and 5 - 5 Fr), and in In an immunohistochemical study with antibodies to prostaglandin E2 receptors, the values of the expression index were 1.5 or more conventional units. In 14 children, during probing, the ductus arteriosus was impassable for polyurethane central venous or umbilical catheters ranging in size from 1 to 5 Fr, and the values of the expression index in immunohistochemical studies with antibodies to prostaglandin E2 receptors were less than 1.5 conventional units.

The method is carried out as follows: in dead premature babies with a birth weight of less than 1500 g and a gestational age of 22-32 weeks, an arterial duct is isolated during autopsy. The material is fixed in 10% buffered formalin for 12-24 hours and standard histological wiring is performed. Immunohistochemical examination is carried out on paraffin sections, 4-5 microns thick. After deparaffination of micropreparations, the antigen is unmasked by boiling in citrate buffer for 15 minutes in a microwave at a power of 650 W. Endogenous peroxidase block 1% hydrogen peroxide solution with sequential incubation in a humid chamber with primary antibodies. Rabbit polyclonal antibodies to prostaglandin E2 receptors of the IgG class (ThermoFisher Scientific, USA, working dilution 1:200) were used as primary antibodies. Sections are washed with three changes of phosphate buffer between incubations. Visualization of antigen-antibody complexes was carried out using the UltraVision Quanto Detection System HRP (ThermoFisher Scientific, USA). In order to exclude the possibility of obtaining false-negative and false-positive results, each immunohistochemical study is carried out with the formulation of a positive and negative control. The negative control excluded specific staining of preparations. The positive control confirmed the specificity of the immunohistochemical reaction with these antibodies.

The results of the immunohistochemical reaction are evaluated in 10 fields of view of the microscope in 100 cells at a magnification of x 400. For quantitative assessment, the expression index of the prostaglandin E2 receptor in the wall of the arterial duct was determined by the formula:

Expression index = ∑P(i) ⋅ i/100 conventional units,

where i is the intensity of staining of the immunohistochemical reaction of sections of the arterial duct, assessed in 10 fields of view of the microscope in 100 cells of the preparation at a magnification of x 400, expressed in points from 0 to 3;

P(i) is the percentage of cells stained with different intensities.

[Pathology of the Breast / F.A. Tavassoli et al. - Stamford: Appleton-Lange, 1999].

If the expression index of the prostaglandin E2 receptor is equal to or more than 1.5 conventional units, a functioning ductus arteriosus is diagnosed, and if the value is less than 1.5 conventional units, a non-functioning ductus arteriosus is diagnosed.

The novelty of the invention lies in the fact that for the first time, a morphological diagnosis of a functioning ductus arteriosus in very premature newborns is proposed by determining the expression of the prostaglandin E2 receptor in the wall of the ductus arteriosus.

Functional closure of the duct is achieved by contraction of smooth muscles due to regulatory factors of vasoreactivity, which include prostaglandin E2 (PGE2) [Hung Y.C., Yeh J.L., Hsu J.H. Molecular Mechanisms for Regulating Postnatal Ductus Arteriosus Closure // Int J Mol Sci - 2018. - Vol.19, No. 7. – P.1861]. The latter, through intracellular cAMP, reduces the sensitivity of vascular wall myocytes to the effects of calcium ions [Yokoyama U. Prostaglandin E-mediated molecular mechanisms driving remodeling of the ductus arteriosus // Pediatr Int. - 2015. - Vol. 57, No. 5. - P. 820-827].

The available literature lacks data on the use of the immunohistochemical index of prostaglandin E2 receptor expression as a marker of a functioning ductus arteriosus in very preterm infants with a birth weight of less than 1500 g.

Distinctive features of the method:

determine the expression index of the prostaglandin E2 receptor in the wall of the arterial duct according to the formula:

Expression index = ∑P(i) ⋅ i/100 conventional units,

where i is the intensity of staining of the immunohistochemical reaction of sections of the arterial duct, assessed in 10 fields of view of the microscope in 100 cells of the preparation at a magnification of x 400, expressed in points from 0 to 3;

Р(i) – percentage of cells stained with different intensity,

and if the value of the expression index of the prostaglandin E2 receptor is equal to or more than 1.5 conventional units, a functioning ductus arteriosus is diagnosed, and if the value is less than 1.5 conventional units, a non-functioning ductus arteriosus

The essence of the proposed method is illustrated by the following examples.

Example 1 . Child M. was born from 1 pregnancy 1 premature operative delivery at 25 weeks of gestation. Pregnancy was complicated by severe preeclampsia, placental insufficiency, chronic intrauterine fetal hypoxia. Birth weight 650 g, height 30 cm. Apgar score 5/6 points. On the 2nd day, an echocardiographic study was performed and a functioning arterial duct with a diameter of 2 mm was diagnosed. The child died on the 3rd day of life from a bilateral non-traumatic intraventricular hemorrhage of the III degree. At autopsy, the ductus arteriosus is passed for a bellied probe, the diameter of the duct is 2 mm.

According to the proposed method, the expression index of the prostaglandin E2 receptor in the wall of the arterial duct was determined, which was 1.7 conventional units, which corresponds to a functioning arterial duct.

Conclusion: functioning ductus arteriosus.

Example 2 . Newborn girl N., from 3 pregnancies 2 premature operative birth at 30 weeks. The course of pregnancy was complicated by moderate preeclampsia, anemia stage I, placental insufficiency, chronic intrauterine fetal hypoxia. Birth weight 1100 g, height 39 cm. On the 2nd day, an echocardiographic study was performed, FAP was not visualized. The child died on the 5th day of life from bilateral interstitial pneumonia of unspecified etiology. At autopsy, the ductus arteriosus was impassable for a bellied probe and passable for a polyurethane central venous catheter with a diameter of 0.8 mm.

According to the proposed method, the index of expression of the prostaglandin E2 receptor in the wall of the arterial duct was determined, which was 1.5 conventional units, which corresponds to a functioning arterial duct.

Conclusion: functioning ductus arteriosus.

Example 3 . Child S. was born from 3 pregnancies 2 premature operative birth at 31 weeks of gestation. Pregnancy was complicated by isthmic-cervical insufficiency, placental insufficiency, chronic intrauterine fetal hypoxia. Birth weight 1450 g, height 42 cm. On the 2nd day, an echocardiographic study was performed, a functioning arterial duct with a diameter of 1.5 mm was diagnosed. The child died on the 9th day of life from bilateral interstitial pneumonia. At autopsy, the ductus arteriosus for the bulbous probe and polyurethane central venous and umbilical catheters is not passable.

According to the proposed method, the expression index of the prostaglandin E2 receptor in the wall of the ductus arteriosus was determined, which was 0.9 conventional units, which corresponds to a non-functioning ductus arteriosus.

Conclusion: non-functioning ductus arteriosus.

The advantages of the proposed method.

1. It is an objective, highly informative and reliable method of morphological diagnostics, allowing at the molecular level to assess the functioning of the arterial duct in very premature newborns.

2. The method improves the accuracy of diagnosis, the quality of the histological conclusion and the completeness of the clinical and morphological comparison of the results of the study of the cardiovascular system in very premature newborns.

3. The method is easy to evaluate the results; its implementation does not require expensive equipment.

Claims (5)

A method for post-mortem morphological diagnosis of a functioning ductus arteriosus in very premature newborns with a birth weight of less than 1500 g by examining biological material, characterized in that the expression index of the prostaglandin E2 receptor in the arterial duct wall is determined by the formula: Expression index = ∑P(i) ⋅ i/100 conventional units, where i is the intensity of staining of the immunohistochemical reaction of sections of the arterial duct, assessed in 10 fields of view of the microscope in 100 cells of the preparation at a magnification of x 400, expressed in points from 0 to 3; Р(i) – percentage of cells stained with different intensity, and if the value of the expression index of the prostaglandin E2 receptor is equal to or more than 1.5 conventional units, a functioning ductus arteriosus is diagnosed, and if the value is less than 1.5 conventional units, a non-functioning ductus arteriosus is diagnosed.