RU2441240C1 - Diagnostic technique for axonal demyelinating polyneuropathies - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: stage of plasma protein fractionation is conducted with using magnetic microparticles. It is followed by mass spectrometric analysis of an eluate sample. The derived mass spectra are compared for the presence or absence of a number of peaks matched with biomarkers of axonal demyelinating polyneuropathies, namely Guillain-Barre syndrome and chronic inflammatory demyelinating polyneuropathy. The absence of characteristic numbers of the mass spectra peaks testifies to the absence of axonal demyelinating polyneuropathy in a patient. ^ EFFECT: use of the technique allows higher diagnostic accuracy in axonal demyelinating polyneuropathies. ^ 6 tbl, 6 dwg, 2 ex

Description

The invention relates to medicine, namely to neurology, infectious diseases, laboratory diagnostics, and for the diagnosis of demyelinating diseases of the peripheral nervous system, such as Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy.

Axonal demyelinating polyneuropathies are one of the most serious and pressing problems of modern medicine, both in Russia and around the world. Currently, there are two groups of acquired autoimmune axonal demyelinating polyneuropathies, which include Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) [1].

Different approaches to the treatment of patients with GBS and HVDP require timely differential diagnosis of these diseases, which in some cases causes great difficulties, especially in the early stages of the disease, when it is difficult to recognize and predict the further type of autoimmune process in patients with LDL [2, 3]. It is important to note that to date there are no clear differential diagnostic criteria for various forms of GBS and HVDP [4]. As a result of incorrect diagnosis, patients do not receive adequate pathogenetic therapy, which leads to increased disability and a deterioration in the quality of life of patients [2, 5].

A known method for the diagnosis of Guillain-Barré syndrome, which consists in assessing the totality of clinical and laboratory criteria developed by WHO specialists in 1993 (table 1) [6]. This diagnostic method is used in conjunction with a method based on electroneuromyography data (table 2), which allows differentiating different forms of Guillain-Barré syndrome with each other.

Table 1 Diagnostic Criteria for Guillain-Barré Syndrome Feature Category Signs Signs needed to diagnose GBS 1. Progressive muscle weakness more than in one limb. 2. Tendon areflexia. Symptoms supporting the diagnosis of GBS A. Clinical signs 1. Progression: symptoms and signs of motor impairment (muscle weakness) develop rapidly, but stop growing by the end of the 4th week from the onset of the disease. 2. The relative symmetry of the lesion. 3. Sensory impairment. 4. Characteristic damage to the cranial nerves, paresis of the facial muscles. 5. Recovery usually begins 2-4 weeks after the cessation of disease progression, but may be delayed for several months. Most patients are characterized by a complete restoration of the functions of the nervous system. 6. Vegetative disorders: tachycardia, arrhythmias, postural arterial hypotension, arterial hypertension, vasomotor disorders. 7. Lack of fever at the onset of the disease (fever at the onset of the disease in a small number of patients may be associated with intercurrent infections). Fever does not exclude GBS, but raises the question of the possibility of another disease, in particular polio. B. Options 1. Severe sensory impairment with a pain component.
2. Progression for more than 4 weeks, relapses are possible.
3. The cessation of progression without subsequent recovery or the preservation of severe persistent residual symptoms.
4. Functions of the sphincters: usually the sphincters are not affected, but in some cases, urination disorders are possible.
5. Damage to the central nervous system: there is no reliable evidence of the possibility of damage to the central nervous system. Some patients may have deep cerebellar ataxia, pathological extensor type stop signs, dysarthria, or a fuzzy level of sensory disturbances, which does not exclude the diagnosis of GBS if other typical clinical symptoms are present. Symptoms supporting the diagnosis of GBS C. Changes in cerebrospinal fluid 1. Protein: a week after the onset of the disease, the concentration of protein in CSF becomes elevated.
2. Cytosis: the content of mononuclear leukocytes in CSF up to 10 in 1 μl. With a content of 1 μl CSF of 20 or more leukocytes, a thorough examination is necessary. If their content is 50 or more in 1 μl, the diagnosis of GBS is rejected. Signs of doubt in the diagnosis 1. Pronounced persistent asymmetry of motor disorders.
2. Persistent pelvic disorders.
3. The presence of pelvic disorders in the onset of the disease.
4. The content of mononuclear leukocytes in the CSF is more than 50 in 1 μl.
5. The presence in the CSF of polymorphic nuclear leukocytes.
6. A clear level of sensitivity disorders. Signs precluding diagnosis 1. Abuse of volatile organic solvents.
2. Disorders of porphyrin metabolism, implying a diagnosis of acute intermittent porphyria.
3. Recently transferred diphtheria.
4. The presence of symptoms of neuropathy due to proven lead intoxication (paresis of the muscles of the upper limb, sometimes asymmetric, with severe weakness of the extensors of the hand).
5. The presence of exclusively sensory disturbances (however, some authors consider acute sensory neuropathy, which manifests itself exclusively as a violation of sensitivity) as a casuistically rare atypical form of GBS.
6. A reliable diagnosis of another disease that manifests itself with symptoms similar to GBS (poliomyelitis, botulism, toxic polyneuropathy).

table 2 Electroneuromyographic (ENMG) signs of various forms of Guillain-Barré syndrome Diagnostic value of signs Signs Symptoms characteristic of acute inflammatory demyelinating polyneuropathy 3 out of 4 signs are required: 1) Decreased conduction speed for two or more motor nerves to: a) <80% of the lower limit of the norm (with the amplitude of the M-response> 80% of the lower limit of the norm); b) <70% of the lower limit of the norm (with the amplitude of the M-response <80% of the lower limit of the norm). 2) Partial blocking or abnormal temporal dispersion - in at least one motor nerve. 3) Elongation of distal latency in more than two nerves to: a)> 125% of the upper limit of the norm (with the amplitude of the M-response> 80% of the lower limit of the norm); b)> 150% of the upper limit of the norm (with an amplitude of the M-response <80% of the lower limit of the norm); 4) No F-wave or minimum lengthening
F-wave latency in two or more nerves up to: a)> 120% of the upper limit of the norm (with the amplitude of the M-response> 80% of the lower limit of the norm); b)> 150% of the upper limit of the norm (with an amplitude of the M-response <80% of the lower limit of the norm). Symptoms characteristic of acute motor axonal neuropathy 1. None of the above signs of demyelination in any nerve (excluding any one sign in 1 nerve, if the amplitude of the M-response is <10% of the lower limit of the norm).
2. The amplitude of the M-response <80% of the lower limit of the norm (not less than 2 nerves).
3. Decreased sensory response or unexcited motor and sensory nerves. Symptoms characteristic of acute motor-sensory axonal neuropathy 1. None of the above signs of demyelination in any nerve (excluding any one symptom in 1 nerve, if the amplitude of the M-response is <10% of the lower limit of the norm).
2. The amplitude of the M-response <80% of the lower limit of the norm (not less than 2 nerves).
3. Excitable motor nerves.

The disadvantage of the above methods is the low accuracy with isolated damage to the thin nerve fibers, which is quite common in patients with GBS.

Well-known methods for diagnosing CVD based on criteria developed by the American Academy of Neurology (AAN), the Causes and Treatment Group for Inflammatory Neuropathies (INCAT), and the Saperstein criteria presented in Table 3 can only be used in the diagnosis of CVD with a typical course. It is impossible to distinguish acute-onset HDV and Guillain-Barré syndrome in the first 4 weeks of the disease, which entails the selection of the wrong patient management tactics and, as a result, the development of an adverse outcome.

Thus, despite the existing methods of diagnosis, even with the undoubted clinical signs of polyneuropathy, it is extremely difficult to establish the correct nosological diagnosis, and with an atypical course of the disease it is impossible even with the most thorough investigation, and it is possible only with further progression of the disease [2].

Given the autoimmune nature of the development of axonal demyelinating polyneuropathies, an important place in their diagnosis is occupied by immunological research methods. However, the use of proteomic technologies seems to be the most promising, since the latter do not require orientation to the specificity of the antigen-antibody interaction, which in autoimmune processes fails due to the presence of multiple “cross” nonspecific reactions [7]. A systematic approach based on the detection of various pathological conditions in the body of biomarkers by proteomic methods, as a rule, requires serious processing of the results using the latest bioinformation technology, which further increases the diagnostic significance of proteomic studies [8].

The medical aspects of proteomics are now in first place. Diagnostic tools for proteomic research, such as SELDI-TOF MS and MALDI-TOF MS technologies, are successfully used to identify biomarkers of various diseases.

The literature describes examples of the successful use of SELDI-TOF MS for the detection of biomarkers, primarily various oncological diseases (cancer of the stomach, colon and colon cancer, hepatocytic carcinoma, ovarian cancer, endometrial cancer, lung cancer, nephroblastoma, thyroid tumors, cancer prostate).

Having a number of advantages in relation to SELDI, MALDI-TOF MS technology is used to determine biomarkers of colorectal cancer, being a very sensitive (95.2%) and specific (90%) diagnostic method for this disease [9]. MALDI analysis is also used for screening studies to form a risk group for the development of gastric cancer [10]; it can be used to assess the benignness or malignancy of breast tumors recorded during mammography [11]. This method determines the biomarkers of nasopharyngeal carcinoma [12]. Evidence is given of the high efficiency of MALDI mass spectrometry in determining biomarkers of stage I ovarian cancer [13]. This method is used to register biomarkers of myocardial infarction (cardiotropins) [14], brain disorders [15].

The success of the topic in diagnosing diseases of the nervous system is much more modest, since they required preliminary decoding of the proteome not only in blood plasma, but also in cerebrospinal fluid in a model of various diseases [16]. As a result, it was found that protein glycosylation is disturbed in such neurodegenerative diseases as Creutzfeldt-Jakob disease, Alzheimer's disease, and Parkinson's disease [17].

A known method for the diagnosis of syphilis using the method of direct proteomic profiling of blood serum [18].

The methods of proteomic analysis were also used for Guillain-Barré syndrome [19, 20]. Experimental studies of cerebrospinal fluid (CSF) in patients with Guillain-Barré syndrome were performed using two-dimensional gel electrophoresis and time-of-flight MALDI-TOF mass spectrometry according to generally accepted standard protocols, followed by detection of abnormal cerebrospinal fluid proteins and measurement of the concentration of these proteins by ELISA according to standard methods . So, Chiang H.L. et al. as a result of applying the above diagnostic method revealed an increase in the level of transthyretin in the cerebrospinal fluid during GBS and concluded that such a reaction can be protective to nerve damage [21]. A Yang Y.R. et al. revealed a decrease in the concentration of cystatin C in CSF in patients with GBS and concluded that this protein may be involved in the pathophysiological process in GBS and requires further study of its role in the pathogenesis of the disease [22]. Being experimental, the results of these studies require further confirmation and cannot serve as a prototype for our research. At the same time, the study of CSF used for diagnosis involves an invasive medical procedure, often associated with technical difficulties, and a lumbar puncture procedure. At the same time, the blood sampling used for our studies can be carried out by paramedical personnel and is a routine procedure.

The MALDI mass spectrometry method used for the diagnosis of other diseases provides for the implementation of standard protocols for fractionation of protein-peptide mixtures, which leads to high requirements for their reproducibility in proteomic analysis of blood serum [23], which are extremely rarely achieved in real clinical practice. In addition, classification models built on the basis of such fractionation do not always have high sensitivity and specificity, which in this case does not allow recommending this method as a diagnostic method.

The use of proteomic technologies in the diagnosis of axonal-demyelinating polyneuropathies (GBS and HVDP) using the advanced protocol for fractioning blood serum of patients with subsequent MALDI mass spectrometry and identification of biomarkers of these diseases will allow them to be verified with high sensitivity and specificity.

The objective of the invention is to improve the accuracy of diagnosis of axonal demyelinating polyneuropathies, namely Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy.

The technical result consists in the identification in the patient's blood serum of sets of mass spectrometric peaks, which are biomarkers of acute and chronic forms of axonal demyelinating polyneuropathy.

This technical result is achieved due to the fact that a patient with suspected axonal demyelinating polyneuropathy is fractionated with protein-peptide mixtures of blood serum samples using magnetic microparticles, while the eluate sample is applied to a polished steel 384-point target in two repetitions and after drying in air, a matrix solution is applied to the sample, represented by a mixture of 2.4 mg of 2,5-dihydroxybenzoic acid and 3 mg of α-cyanohydroxycinnamic acid for every milliliter of methane solution ol: acetonitrile: water, mixed in a ratio of 5: 4: 1; mass spectrometric analysis of the obtained eluate sample for the presence or absence of a set of peaks corresponding to the biomarkers of axonal demyelinating polyneuropathies, the presence of a set of peaks in the mass spectrum of the blood serum characterized by the ratio of molecular weight to charge (m / z): 1945.26; 2787.18; 4,477.75; 3,985.3; 2,843.56; 2,239.51; 11784.46, indicates the presence of Guillain-Barré syndrome in the patient, the presence of a set of peaks characterized by the ratio of molecular mass to charge (m / z): 1945.29; 1886.18; 1490.03; 3,985.32; 1552.5; 3,401.42; 11783.91, indicates that the patient has a chronic inflammatory demyelinating polyneuropathy, and the absence of these sets of peaks in the mass spectrum indicates the absence of axonal demyelinating polyneuropathy in the patient. The use of magnetic particles with a weak cation exchange surface (MB-WBX) and mass spectrometric analysis of the eluate sample using the MALDI-TOF method are preferred. However, it should be understood that other types of microparticles, as well as alternative methods of mass spectrometry, can be used to implement the present invention.

To conduct research on the search for biomarkers specific for GBS or HVDP, 70 patients with axonal demyelinating polyneuropathies were examined. The main group consisted of 40 patients with Guillain-Barré syndrome (of which 20 men and 20 women). The comparison group included 30 patients with chronic inflammatory demyelinating polyneuropathy (of which 14 men and 16 women). The control group consisted of 25 healthy donors (of which 12 were men and 13 were women).

The criteria for inclusion of patients in the main observation groups were: suspicion of axonal demyelinating polyneuropathy (GBS, CVD); age from 16 to 70 years; patient informed consent for examination. Criteria for exclusion of patients from the study were: polio, botulism, diphtheria, HIV infection; myasthenia gravis; toxic polyneuropathy; diabetes; impaired porphyrin metabolism; chronic alcoholism and / or drug addiction; organic lesions of the central nervous system, excluded according to the results of clinical, laboratory and instrumental data, as well as the patient's refusal to study.

Persons included in the control group did not reveal concomitant chronic diseases of the nervous system and at the time of the examination, no subjective and objective signs of acute diseases were detected; a history of no allergic reactions and autoimmune conditions, as well as alcoholism and drug addiction.

Research methodology: 1) sample preparation (fractionation of blood serum proteins); 2) mass spectrometry; 3) analysis of the data.

1. Fractionation of protein-peptide mixtures of blood serum samples was performed on a specialized ClinProt robot (Bruker Daltonics GmbH, Germany) using a profiling kit containing magnetic microparticles with a weak cation exchange surface (MB-WCX), according to the protocol recommended by the manufacturer, with slight modifications consisting in the fact that the eluates were applied to a polished steel 384-point mass spectrometric target in two repetitions, after drying in air, a matrix solution was applied to the sample, representing a mixture of 2.4 mg of 2,5-dihydroxybenzoic acid and 3 mg of α-cyanohydroxycinnamic acid for each milliliter of a solution of methanol: acetonitrile: water, mixed in a ratio of 5: 4: 1.

The conclusion about the preferred use of magnetic microparticles with a weak cation exchange surface (MB-WCX) was made according to the results of our experimental studies of control samples of blood serum (n = 36). For the fractionation of protein-peptide mixtures of blood serum samples, 3 types of magnetic microparticles were used: with reversed-phase (MB-HIC 8, MB-HIC 18), weak cation exchange (MB-WCX) and metal-affinity (MB-IMAC Cu) functionalized surfaces . As a result of comparing the mass spectrometric profiles of blood serum samples previously fractionated on various types of magnetic microparticles, we chose microparticles with a weak cation exchange surface MB-WCX, which ensure registration of the largest number of mass spectrometric peaks compared to other types of particles (figure 1).

Then, the conditions of mass spectrometric analysis of the protein fractions of human blood serum isolated using magnetic microparticles were worked out. For this, from the combination of aromatic compounds recommended for mass spectrometry of protein molecules (Table 4), we tested two matrices: derivatives of benzoic and cinnamic acid - 2,5-dihydroxybenzoic and α-cyano-4-hydroxycinnamic acid.

Table 4 Organic compounds used as the basic substance of the matrix in MALDI mass spectrometry of protein molecules and peptides Connection Name Chemical formula Absorption wavelength (nm) Application area α-cyano-4-hydroxycinnamic acid

337, 355 Proteins and peptides with a mass of less than 10 kDa. Less commonly, lipids and oligonucleotides 3,5-dimethoxy-4-hydroxycinnamic (synapinic) acid

337, 355, 266 Protein molecules weighing more than 10 kDa and glycoproteins 3-methoxy-4-hydroxycinnamic (ferulic) acid

337, 355, 266 High molecular weight protein molecules 3,4-dihydroxycinnamic (caffeic) acid

337, 355, 266 High molecular weight protein molecules 2,5-dihydroxybenzoic acid

337, 355, 266 A wide range of proteins and peptides, including phosphopeptides and glycoproteins, as well as lipids and oligosaccharides

Due to the better ionization of small protein molecules (up to 10 kDa) on alpha-4-hydroxycinnamic acid, the use of this matrix alone did not allow to obtain a uniformly rich mass spectrum, a pronounced shift towards small masses was observed. On the other hand, when using 2,5-dihydroxybenzoic acid, a uniform mass spectrum of proteins was observed with good peak resolution. However, the 2,5-dihydroxybenzoic acid was characterized by uneven crystallization on the metal plate, which excluded the automatic accumulation of mass spectra necessary for standardizing the protocol.

A solution was found in the combined use of 2,5-dihydroxybenzoic and α-cyano-4-hydroxycinnamic acids. Such a composite composition of the matrix for ionization ensured the uniformity of the accumulated mass spectra, on the one hand, and made it possible to form homogeneous crystals suitable for automatic measurement with the analyte.

Modification of the standard fractionation protocol made it possible to significantly increase the number of mass spectrometric peaks detected in the eluates, as well as the quality and reproducibility of the obtained mass spectrometric profiles, as illustrated by figures 2 and 3. Figure 2 shows the average mass spectrometric profiles of blood serum samples after fractionation with using magnetic microparticles MB-WCX. The figure shows that the number of detected components in peptide-protein mixtures obtained after fractionation of blood serum using a modified separation protocol (A) significantly exceeds the number of components in eluates obtained by fractionation of plasma and blood serum using standard protocol (B), especially clearly these differences are observed in the mass range up to 4 kDa (figure 3).

2. Mass spectrometric analysis was performed using an Ultraflex time-of-flight MALDI-TOF mass spectrometer (Bruker Daltonics GmbH, Germany). The samples were desorbed by irradiation with a nitrogen laser (wavelength 337 nm) operating at a frequency of 25 Hz. To remove the matrix peaks, a maximum signal suppression level of up to 900 Da was used. Automatic registration of mass spectra was carried out in the linear mode of positively charged ions in the mass range 1-15 kDa. For calibration, a calibration mixture containing peptides and proteins in the mass range of 1-17 kDa was used. To increase the sensitivity of mass spectrometric detection of proteins and peptides, the excess matrix was removed with 10 laser pulses at a power of 45%, followed by accumulation of data at a laser power of 27%. For each spectrum, the results of 720 laser pulses were summarized (60 pulses from 12 different spot points). Spectra with a signal-to-noise ratio> 5 and a resolution> 300 were summarized. To control the mass spectrometer, including setting operating modes and recording mass spectra, we used the FlexControl 2.4 software package (Bruker Daltonics, Germany). In figures 4-6 in the form of pseudogels shows mass spectrometric profiles of the compared groups of blood serum samples of patients with GBS and practically healthy donors, patients with CIDP and practically healthy donors, as well as patients with CIDP and with GBS, respectively. As a result of integration of the mass spectrometric profiles of the above comparison pairs using the ClinProTools 2.1 program, 219, 222 and 236 peaks were respectively marked.

3. Analysis of mass spectrometric data

Analysis of the obtained data was carried out using the ClinProTools version 2.1 mass spectrometric analysis software package (Bruker Daltonics, Germany), which meets all the main requirements for the search for biomarkers in complex biological mixtures and their validation. The ClinProTools bioinformatics system (version 2.1) implements two methods for constructing classification models (classifiers) for analyzing the distribution of biomarkers between groups: a genetic algorithm and a controlled neural network, which are generally recognized and effective methods of mathematical analysis of mass spectrometric data for solving problems associated with the construction of classifiers . The determination of biomarkers detected in models using these algorithms as mass spectrometric signals is carried out by selecting the optimal parameters of the algorithms: the number of specified mass spectrometric signals (peaks) and the number of "neighbors" (for both algorithms), the number of iterations (for the genetic algorithm) to achieve the highest cross-validation and recognition capabilities.

As a result of mass spectrometric analysis of protein-peptide mixtures of blood serum of patients with GBS using the genetic algorithm, 7 significant mass spectrometric peaks were determined with the following average values of the ratio of molecular weight to charge (m / z): 1945.26; 2787.18; 4,477.75; 3,985.3; 2,843.56; 2,239.51; 11784.46, which were included in the classification model and identified as biomarkers of GBS (table 5).

Table 5 The values of m / z peaks included in the classification model of blood serum "GBS control", built using the genetic algorithm Index Mass Start mass End mass Weight 34 1945 1937 1950 2.006001001 60 2787 2780 2796 1.787940192 102 4478 4463 4482 1.349909864 87 3985 3976 3990 0.928244527 62 2844 2834 2851 0.738832543 45 2240 2234 2245 0.678443885 219 11784 11774 11797 0.266230980 Index - peak serial number
Mass - m / z peak value
Start Mass, End Mass - m / z values corresponding to the beginning and end of the mass spectrometric peak, respectively
Weight - statistical peak weight corresponding to the numerical value of the coefficient in the algebraic expression of the classification model

As a result of mass spectrometric analysis of protein-peptide mixtures of blood serum of patients with CIDP using the genetic algorithm, 7 significant mass spectrometric peaks were determined with the following average values of the ratio of molecular weight to charge (m / z): 1945.29; 1886.18; 1490.03; 3,985.32; 1552.5; 3,401.42; 11,783.91, which were included in the classification model and are defined as biovarkers of HVDP (table 6).

Table 6 Values of m / z peaks included in the classification model of blood serum "HVDP control", constructed using the genetic algorithm Index Mass Start mass End mass Weight 34 1945 1940 1950 2,051317454 32 1886 1883 1891 1.923997493 19 1490 1486 1497 1,749895004 86 3985 3975 3989 1.454915710 23 1553 1549 1558 1,090828045 77 3401 3399 3412 1,082192700 222 11784 11768 11797 0.265484282 Index - peak serial number Mass - m / z peak value Start Mass, End Mass - m / z values corresponding to the beginning and end of the mass spectrometric peak, respectively Weight - statistical peak weight corresponding to the numerical value of the coefficient in the algebraic expression of the classification model

A comparative analysis of the obtained mass spectrometric data was carried out using the computer program ClinProTools 2.1 (Bruker Daltonics, Germany) using a mathematical genetic algorithm that allowed us to identify characteristic sets of peaks, the consistent change in their areas with high specificity and sensitivity differentiated mass spectrometric profiles of patient blood serum with LDL and practically healthy donors (for GBS, the values of these characteristics reached 100% and 100%, and for HVDP 94.1% and 100%, respectively).

Analysis of mass spectrometric profiles of blood serum samples obtained from patients with GBS and HVDP, allowed us to build a classification model that distinguishes these pathologies from each other, with a specificity of 88.9% and a sensitivity of 80%.

Thus, the presence in the mass spectrum of the blood serum of the patient a set of peaks with the following values of the ratio of molecular weight to charge: 1945.26; 2787.18; 4,477.75; 3,985.3; 2,843.56; 2,239.51; 11784.46, indicates the presence of Guillain-Barré syndrome in the patient, the presence of peaks with the following values of the ratio of molecular weight to charge: 1945.29; 1886.18; 1490.03; 3,985.32; 1552.5; 3,401.42; 11783.91, indicates the presence of chronic inflammatory polyneuropathy in the patient, and the absence of these sets of peaks in the mass spectrum of blood serum indicates the absence of axonal demyelinating polyneuropathy in the patient.

The invention is illustrated by the following graphic materials.

Figure 1. Averaged mass spectra of blood serum samples obtained from practically healthy donors after their fractionation on magnetic microparticles MB-HIC8 (A), MB-WCX (B) and MB-IMAC Cu (C) using a modified protocol.

Figure 2. Average mass spectrometric profiles of blood serum samples fractionated using magnetic microparticles with a weak cation exchange surface according to modified (A) and standard (B) protocols.

Figure 3. Average mass spectrometric profiles of serum samples fractionated on MB-WCX magnetic particles using modified (A) and standard (B) protocols in the m / z range from 1500 to 3500 Da.

Figure 4. Averaged mass spectrometric profiles of blood serum samples of healthy donors (a) and patients with GBS (b). Fractionation of the samples was carried out using magnetic microparticles MB-WCX. The ordinate shows the normalized intensity of the mass spectrometric peaks, and the abscissa represents the mass to charge ratio. The intensity of the staining of the bands corresponds to the intensity of the peak in the spectrum. Data are shown as pseudogel. The inset at the top shows the averaged mass spectra of the most significant peaks included in the classification model constructed using the genetic algorithm.

Figure 5. Averaged mass spectrometric profiles of blood serum samples of healthy donors (a) and patients with CIDP (b). Fractionation of the samples was carried out using magnetic microparticles MB-WCX. The ordinate shows the normalized intensity of the mass spectrometric peaks, and the abscissa represents the mass to charge ratio. The intensity of the staining of the bands corresponds to the intensity of the peak in the spectrum. Data are shown as pseudogel. The inset at the top shows the averaged mass spectra of the most significant peaks included in the classification model constructed using the genetic algorithm.

6. Averaged mass spectrometric profiles of blood serum samples of patients with CIDP (a) and GBS (b). Fractionation of the samples was carried out using magnetic microparticles MB-WCX. The ordinate shows the normalized intensity of the mass spectrometric peaks, and the abscissa represents the mass to charge ratio. The intensity of the staining of the bands corresponds to the intensity of the peak in the spectrum. Data are shown as pseudogel. The inset at the top shows the averaged mass spectra of the most significant peaks included in the classification model constructed using the genetic algorithm.

The method is as follows:

in a patient with suspected axonal demyelinating polyneuropathy, a direct proteomic profiling of a blood serum sample is performed to identify biomarkers of demyelinating polyneuropathy (GBS or HVDP), which includes: 1) fractionation of protein-peptide mixtures of blood serum samples using magnetic standard microparticles with a weak surface microparticle exchange a specialized robot (e.g., ClinProt, Bruker Daltonics GmbH, Germany), while eluates are applied to a polished steel 384-point ith mass spectrometric target in two repetitions, after drying in air, the sample is coated with a solution of a matrix represented by a mixture of 2.4 mg of 2,5-dihydroxybenzoic acid and 3 mg of α-cyanohydroxycinnamic acid for every milliliter of a solution of methanol: acetonitrile: water, mixed in 5: 4: 1 ratio; 2) mass spectrometric analysis of the obtained eluate sample using a time-of-flight MALDI-TOF mass spectrometer (for example, Ultraflex, Bruker Daltonics GmbH, Germany); 3) a comparative analysis of the obtained mass spectra is carried out using a software package for processing mass spectrometric analysis data (for example, ClinProTools 2.1, Bruker Daltonics, Germany) and in the presence of a set of discriminant peaks in the mass spectrum of blood serum with the following values of the ratio of molecular weight to charge (m / z): 1945.26; 2787.18; 4,477.75; 3,985.3; 2,843.56; 2,239.51; 11784.46, the patient is diagnosed with Guillain-Barré syndrome in the patient, in the presence of discriminant peaks with the following values of the ratio of molecular weight to charge (m / z): 1945.29; 1886.18; 1490.03; 3,985.32; 1552.5; 3,401.42; 11783.91, the patient is diagnosed with chronic inflammatory polyneuropathy, and in the absence of these sets of peaks in the mass spectrum of blood serum, the patient has the axonal demyelinating polyneuropathy rejected.

The proposed method with high specificity and sensitivity allows us to differentiate Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy and can be introduced into the practice of neurological, infectious and multidisciplinary clinics in collaboration with research institutes that have a proteomic research laboratory.

Clinical example 1

Patient N., 46 years old, was admitted to the NCH RAMS on the 9th day of illness with complaints of weakness in the arms and legs, pain in the calf muscles, slight numbness of the hands and feet. On examination, tetraparesis was observed with a decrease in strength in the proximal sections of the hands to 3 points, in the distal sections to 2 points, in the proximal sections of the legs to 3 points, there were no active movements in the feet. Violation of deep sensitivity was manifested by a decrease in vibration sensitivity in the fingers and toes. The absence of deep tendon reflexes, increased sweating and symptoms of damage to the V, VII, IX, XI and XII cranial nerves in the form of a decrease in the strength of the masticatory and facial muscles of the face, neck and tongue muscles, speech and swallowing disorders were also noted.

From the anamnesis: two weeks before the development of neurological symptoms, she suffered an acute respiratory disease with an increase in body temperature to 37.5 ° C and a sore throat when swallowing. Against the background of symptomatic therapy, my health returned to normal within 3 days. 10 days after the acute respiratory infections, numbness appeared in the feet, later in the hands. On the 3rd day of the appearance of neurological symptoms, weakness developed in the upper and lower extremities, which slowly increased. On the 8th day I could not walk.

Denies chronic diseases (including diabetes, alcoholism and drug addiction).

When examining changes in clinical and biochemical blood tests and clinical analysis of urine was not detected. Porfobilinogen in urine is not detected.

Antibodies to HIV, hepatitis C virus and HBsAg in blood serum by ELISA were not detected. In the study of blood serum, antibodies to Asialo-GM ₁ and GM _{1 were} detected by ELISA.

When ENMG on the 9th day of the disease revealed a primary demyelinating process in the peripheral nerves of the upper and lower extremities.

MRI showed no signs of organic damage to the central nervous system.

The patient was suspected of acute axonal demyelinating polyneuropathy (GBS). The patient agreed to a blood test by mass spectrometric analysis.

Blood serum was fractionated on a specialized ClinProt robot (Bruker Daltonics GmbH, Germany) using weak cation exchange magnetic particles (MB-WCX), eluates were applied to a polished steel 384-point mass spectrometric target in duplicate, after drying in air on a sample applied a matrix solution represented by a mixture of 2.4 mg of 2,5-dihydroxybenzoic acid and 3 mg of α-cyanohydroxycinnamic acid for every milliliter of a solution of methanol: acetonitrile: water, mixed in a ratio of 5: 4: 1. As a result of the analysis of the obtained mass spectra using the Ultraflex time-of-flight MALDI mass spectrometer (Bruker Daltonics GmbH, Germany), a set of mass spectrometric peaks was revealed, characterized by the following values of the molecular weight to charge ratio (m / z): 1945.26; 2787.18; 4,477.75; 3,985.3; 2,843.56; 2,239.51; 11784.46, which indicates the presence of Guillain-Barré syndrome in the patient.

Observation of the patient in the follow-up for 1 year confirmed the diagnosis.

Clinical example 2

Patient B., 32 years old, was admitted to the NCH RAMS on the 3rd day of illness with a diagnosis of Guillain-Barré syndrome with the development of gross flaccid tetraparesis, bulbar syndrome and respiratory failure, with complaints of weakness in the arms and legs, speech impairment. Examination revealed flaccid tetraparesis with a decrease in strength in the proximal arms and legs to 1 point, in the distal to 3 points. Violation of deep sensitivity was observed in the form of a decrease in vibration sensitivity and joint-muscle feeling at the fingertips of the hands and feet. Pain sensitivity is not impaired. Breathing is independent.

From the anamnesis: 2 weeks before the development of neurological symptoms, she suffered an acute respiratory disease with catarrhal phenomena and an increase in body temperature to 40 ° C. After which there was weakness in the upper and lower extremities, difficulty walking, speech became chanted. Weakness in the limbs increased, joined by the weakness of the muscles of the neck, back and abdomen. On the 4th day of neurological symptoms, she could not get out of bed. In the ward, the patient's condition continued to worsen: weakness in the limbs increased up to plegia, difficulty breathing, and therefore the patient was intubated with trachea and connected to an artificial lung ventilation apparatus.

Denies chronic diseases (including diabetes, alcoholism and drug addiction).

When examining changes in clinical and biochemical blood tests and clinical analysis of urine was not detected. Porfobilinogen in urine is not detected.

Antibodies to HIV, markers of hepatitis A, B and C in blood serum by ELISA were not detected.

Radiography of the chest, paranasal sinuses and cervical spine revealed no pathology. An ECG showed sinus tachycardia.

In the study of cerebrospinal fluid: cytosis - 6/3; lymphocytes - 5; neutrophils - 1; protein - 0.33 g / l. Sowing cerebrospinal fluid did not give growth.

When examining blood serum by ELISA, antibodies to gangliosides were not detected.

When ENMG on the 4th day of the disease revealed a primary demyelinating process in the peripheral nerves of the upper and lower extremities.

MRI showed no signs of organic damage to the central nervous system.

The patient was suspected of acute axonal demyelinating polyneuropathy (GBS). The patient agreed to a blood test by mass spectrometric analysis.

Blood serum was fractionated on a specialized ClinProt robot (Bruker Daltonics GmbH, Germany) using weak cation exchange magnetic particles (MB-WCX), eluates were applied to a polished steel 384-point mass spectrometric target in duplicate, after drying in air on a sample applied a matrix solution represented by a mixture of 2.4 mg of 2,5-dihydroxybenzoic acid and 3 mg of α-cyanohydroxycinnamic acid for every milliliter of a solution of methanol: acetonitrile: water, mixed in a ratio of 5: 4: 1. As a result of the analysis of the obtained mass spectra using an Ultraflex time-of-flight MALDI mass spectrometer (Bruker Daltonics GmbH, Germany), a set of mass spectrometric peaks was revealed, characterized by the following values of the molecular weight to charge ratio (m / z): 1945.29; 1886.18; 1490.03; 3,985.32; 1552.5; 3,401.42; 11783.91, which indicates that the patient has a chronic inflammatory demyelinating polyneuropathy.

Observation of the patient for 2 years confirmed the diagnosis.

Literature

Claims (3)

1. A method for the diagnosis of axonal demyelinating polyneuropathies by direct dark blood profiling of blood serum based on the detection of axon demyelinating polyneuropathies in a serum sample of biomarkers, which includes: 1) fractionation of protein-peptide mixtures of blood serum using magnetic microparticles with application of the eluate to polished steel polished 384 mass spectrometric target in duplicate, after drying in air, a solution of a matrix represented by a mixture of 2.4 is applied to the sample mg of 2,5-dihydroxybenzoic acid and 3 mg of α-cyanohydroxycinnamic acid for every milliliter of a solution of methanol: acetonitrile: water, mixed in a ratio of 5: 4: 1; 2) mass spectrometric analysis of the obtained eluate sample; 3) a comparative analysis of the obtained mass spectra using a software package for processing mass spectrometric analysis data for the presence or absence of a set of peaks corresponding to biomarkers of axonal demyelinating polyneuropathies, and in the presence of a set of peaks in the blood serum mass spectrum with the following values of the molecular ratio mass to charge (m / z): 1945.26; 2787.18; 4,477.75; 3,985.3; 2,843.56; 2,239.51; 11784.46, the patient is diagnosed with Guillain-Barré syndrome, in the presence of peaks with the following values of the ratio of molecular weight to charge (m / z): 1945.29; 1886.18; 1490.03; 3,985.32; 1552.5; 3,401.42; 11783.91, the patient is diagnosed with chronic inflammatory polyneuropathy, and in the absence of these sets of peaks in the mass spectrum of blood serum, the patient has the axonal demyelinating polyneuropathy rejected. 2. The method according to claim 1, wherein the magnetic microparticles are MB-WCX with a weak cation exchange surface. 3. The method according to claim 1, wherein the mass spectrometric analysis of the eluate sample is carried out according to the MALDI-TOF method.

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