RU2629388C2 - Method for sampling of newborns exhaled air condensate during artificial ventilation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for sampling of exhaled air condensate (EAC) of newborns on artificial ventilation consists in application of a condensate collection system and an auxiliary measuring module to monitor the collection parameters. The condensate collection system contains two coaxial cylinders made of glass and having separate outlets on tops, this system being thermostatted with a coolant in the form of a pre-cooled liquid filled in a thermos-type Dewar bottle. During the EAC sample collection procedure, coolant temperature is continuously measured by means of an auxiliary measuring module, represented by a digital thermometer with a thermocouple temperature sensor with a function of temperature readings storage at a given periodicity, and with a chronometer function.

EFFECT: invention allows non-invasive and safe sampling of the exhaled air condensate in newborns under artificial ventilation for further analysis, without introducing any changes to the ventilation mode.

4 dwg, 3 tbl, 5 ex

Description

The invention relates to medicine, namely to neonatology, to methods for collecting condensate of exhaled air of newborns who are under mechanical ventilation in order to monitor the condition of the patient.

In recent years, the analysis of volatile and non-volatile components in exhaled breath condensate (HFC) has become a new tool for non-invasive study of oxidative markers and mediators of inflammation in the airways [1]. This method has been successfully used in studies of oxidative stress markers in children with bronchial asthma [2]. To assess oxidative stress and airway inflammation in newborns, a standard step is to take a sample using tracheal aspiration. However, this is a very invasive procedure for routine use in newborns, especially those who were born prematurely [3]. Correlation of antioxidant status between samples obtained using tracheal aspiration and samples of CVE was shown only very recently [4]. Currently, studies on assessing the condition of newborns using CVI are only just beginning to be carried out and are of great interest due to the non-invasiveness of the procedure for collecting CVV.

As a prototype of the proposed method for the analysis of the proteomic composition of the condensate of the expired air of newborns undergoing artificial ventilation, we used the method described in [4], according to which the selection of newborns' HVC samples was carried out by including the HVC collection system (Rtube, USA) in the output tract of the artificial ventilation of the lungs. The disadvantage of this method is the need to adapt a standardized tube for adult patients Rtube. However, the authors do not indicate whether they used in their final design such a standard Rtube component as an exhalation valve, such as a goose beak. It is known that the outlet valve - the "goose beak" - creates additional resistance and requires the creation of excess pressure, which in the case of collection of HVC in infants who are on artificial ventilation, is an additional risk factor and requires special reservation. The Rtube instructions for use for adult patients separately specify a possible blockage of the “goose beak” valve and it is recommended that each time, before use, a visual check is made of the valve opening freely. The second, not so significant drawback of the Rtube system is the polymer material from which the device is made. This material introduces impurities in the form of polymers during the collection of HVE, which are then easily visible in the chromato-mass spectrometric analysis of the sample. The third significant drawback is the one-time type of Rtube devices - after collecting the sample, the HVV tube is disposed of at a price of $ 100. It is also worth noting that the authors in the collection system itself did not have any auxiliary measuring equipment to control the collection parameters of the HVC - the most important parameter is the condenser cooling temperature.

The task that we are developing a system for collecting EWC for newborns was to eliminate the above disadvantages. The developed device consists of a condensate collection system and an auxiliary measuring module for monitoring the collection parameters, as shown in FIG. 1. The condensate collection system in general is two coaxial cylinders having separate outlets from above (Fig. 2). The outer cylinder is sealed below. Exhaled air is condensed on an external cylinder that is in contact with the refrigerant. The inner cylinder is used to transport air into the device and to increase the path of exhaled air, which increases the efficiency of operation. The refrigerant, as well as its initial temperature, are determined on the basis of the tasks. To increase the stability of the system and increase the duration of the selection, the system can be placed in a heat insulating vessel.

As a temperature measuring module, a digital thermometer with a thermocouple temperature sensor was chosen. The module has the functions of storing temperature readings with a predetermined frequency, a chronometer function and the ability to transfer stored data to a personal computer via a USB port. This makes it possible to register the parameters characterizing the selection of the SEM samples and to take them into account in the further analysis of the obtained samples.

To implement the inventive method for collecting HVS, any typical device for collecting HVS from an inert material, for example, glass, with an appropriate design suitable for adaptation to mechanical ventilation can be used. We selected a concentric glass vessel of a typical production (Lenz Laborglasinstrumente, Wertheim, Germany), a schematic representation of which is shown in FIG. 2. The glass vessel was connected using Teflon tubes to typical ventilators. During the collection procedure, a glass vessel was placed in a thermos with a coolant (refrigerant), pre-cooled to the optimum

temperature (Table 1). Repeated experiments with varying collection times (12–20 min) determined the time required to collect a sufficient amount of condensate (about 1 ml) for further analysis using liquid chromatography – mass spectrometry.

The possibility of realizing the claimed invention with the receipt of the claimed technical result is illustrated by the following examples 1-5 of collection of CVI in newborns on mechanical ventilation using various refrigerants and varying the collection time (Table 1).

CVE was collected from infants on a traditional assisted ventilation, i.e. each respiratory effort of the patient was supported by a hardware breath.

The following types of assisted ventilation were used in the AVEA apparatus:

- ventilation cyclic in time with pressure limitation (TCPL - time cycled pressure limited). This mode is used by default for newborns, the inspiratory flow for newborns is set by default to 8 l / min.

- auxiliary-forced ventilation mode by pressure (Pessure A / C), the inspiratory flow is selected by the device itself in the range of 6-10 l / min,

- auxiliary-forced ventilation mode by volume (Volume A / C), the inspiratory flow is set by default to 8 l / min.

Examples 1-5 show that using the proposed method and device for the collection of HVAC, shown in FIG. 1, it is possible to take a sample of the condensate of exhaled air of the required volume (from 0.5 ml to 1 ml) using various refrigerants and varying the collection time from 10 to 20 minutes. Table 1 shows the experimental conditions using various refrigerants: ice with the addition of salt (NaCl), ice, ethanol (-80 ° C). It is shown that it is best to use ethanol cooled to a low temperature of -80 ° C. When changing the time of collection of the SEM sample from 10 minutes to 20 minutes, it is shown that the optimal time is 20 minutes. Over a period of 20 minutes, it is possible to collect a sample volume of the order of 1.2 ml, which is optimal for further analysis of the molecular composition of the CVB sample using, for example, the method of liquid chromatography-mass spectrometry.

Repeated experiments show (see Example 4-5) that no significant changes in the volume of condensate are observed when good compaction is observed at the connection points of the circuit.

Example 4 shows that using the developed approaches, an analysis of the proteomic composition of exhaled air condensate (BHE) can be performed in 6 newborns who are under artificial ventilation in the intensive care unit. It was shown that within 20 minutes in the mode of mechanical ventilation it is possible to collect the required amount of IQB with a volume of 1.2 ml for further proteomic analysis using chromatography-mass spectrometry. It was possible to identify the characteristic proteins in the composition of the exhaled air of newborns at different stages of development and with various pathologies. In the condensate collected in the regimen with mechanical ventilation, there was a constant spectrum of invariant keratins 1, 10, 2, 9 proteins, which are exogenous proteins, as we showed earlier in the study of adult patients with CVI [5]. Also, non-keratin protein components, the contents of which are variable in different patients, depending on the stage of development and pathology (Table 2, Fig. 3), were identified in the samples of CVI of newborns.

Example 5 shows the possibility of detecting newborns of certain groups of volatile aldehydes, mainly alkanals, 2-alkenals, 4-hydroxy-2-alkenals, which can be metabolites during lipid peroxidation containing polyunsaturated fatty acid residues in collected samples of CVB of newborns (Table. 3, Fig. 4). It was proposed to use their content in biological fluids to assess the level of oxidative stress, i.e. these substances can act as biomarkers of oxidative stress [7, 8].

The possibility of implementing the claimed invention with the receipt of the claimed technical result is demonstrated in examples 1-5. It has been shown that the invention allows efficient sampling of an HVS sample with a volume of at least 1 ml for a period of 20 minutes in infants who are on artificial ventilation without making any changes to the ventilation mode. It is shown in examples 1-5 that the collection system of HVC can be thermostated with a refrigerant in the form of pre-cooled liquid (ethanol, -80 ° C), poured into a Dewar vessel. Examples 1-5 show that this method can be implemented in neonatal units involved in intensive resuscitation, nursing and treatment of premature infants. Examples 4-5 show that this method allows you to non-invasively obtain biomaterial from a patient and conduct proteomic and metabolic analysis of samples to obtain additional information about the condition of patients in order to clarify the diagnosis and treatment tactics.

Thus, the invention allows a non-invasive and safe way to collect the required volume of exhaled breath condensate in infants who are undergoing artificial ventilation, for further analysis of the molecular composition of the IQB in order to obtain additional information about the condition of patients in order to clarify the diagnosis and treatment tactics.

In FIG. 1 shows a diagram of an installation for collecting HVV in a system with connection to an apparatus with artificial lung ventilation (IVL).

In FIG. 2 is a schematic illustration of a concentric glass vessel for collecting an HVS.

In FIG. Figure 3 shows an example of an automated gas chromatography-mass spectrometric analysis of an HF sample. Above is a chromatogram. In the center is the mass spectrum at the time point highlighted in the chromatogram. Bottom: mass spectrum collisively fragmentation (CID) of the peak highlighted in the mass spectrum.

In FIG. Figure 4 shows an example of a mass spectrum of a condensate of exhaled air, (a) an overview mass spectrum, (b) - (g) enlarged mass regions corresponding to individual (b), (c) 2-alkenals, (d) - (g) 4-hydroxy-2-alkenalam.

BIBLIOGRAPHY

1. Filippone M, Bonetto G, Corradi M, et al. Evidence of unexpected oxidative stress in airways of adolescents born very pre-term. Eur Respir J. 2012.40 (5). pp. 1253-9.

2. Ahsman M.J., Tibboel D., Mathot R.A.A., et al. Sample collection, biobanking, and analysis. Handbook of Experimental Pharmacology. 2011.205 pp. 203-217.

3. Marraro GA, Chen C, Piga MA, et al. Acute respiratory distress syndrome in the pediatric age: an update on advanced treatment. Zhongguo Dang Dai Er Ke Za Zhi. 2014.16 (5). pp. 437-47.

4. Rosso MI, Roark S, Taylor E, et al. Exhaled breath condensate in intubated neonates-a window into the lung’s glutathione status. Respiratory Research. 2014.15: 1.

5. V.S. Kurova, A.S. Kononikhin, I.A. Popov et al. Exogenous proteins in the condensate of human exhaled air. Bioorganic chemistry. 2011.V. 37. No. 1. from. 48-52.

6. Hakim, M .; Broza, Y. Y .; Barash, O .; Peled, N .; Phillips, M .; Amann, A .; Haick, H. Chem. Rev. 2012, 112, 5949-5966.

7. Gueraud, F .; Atalay, M .; Bresgen, N .; Cipak, A .; Eckl, P. M .; Huc, L .; Jouanin, I .; Siems, W .; Uchida, K. Free Radical Res. 2010, 44, 1098-1124.

Claims (1)

The method of collecting condensate of exhaled air of newborns who are on mechanical ventilation, characterized in that they use a condensate collection system and an auxiliary measuring module to control the collection parameters, and the condensate collection system contains two coaxial cylinders made of glass and having separate exits from above, this system thermostat the refrigerant in the form of pre-cooled liquid, which is poured into a Dewar type thermos flask, while constantly They measure the temperature of the refrigerant using an auxiliary measuring module, which is used as a digital thermometer with a thermocouple temperature sensor, which has the function of storing temperature readings with a given frequency and the function of the chronometer.

Images