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7Crystallography method for diagnosis of experimental amyloidosis
Yatsenko A., Kushnarev V., Zverev A., Evseenkov V., Antonova I.
Scientific adviser: A.V. Maksimenko
Amur State Medical Academy, Blagoveschensk, Russia
Abstracts: A doctor in his work is facing many diseases and their diagnosis is not made easier, despite the development of diagnostic medicine. Of particular difficulty is the differentiation of various paraproteinemia, one of which is amyloidosis. The disease is characterized by abnormal deposition of amyloid protein in the tissues and organs of the body, while the latter function is disturbed. Therefore, the development of amyloidosis is a method of diagnosing a serious problem for medicine.As such we offer a method of use of crystallography, which is based on the phenomenon of biochemical specificity of biological fluids each individual pathology.
Key words: Crystallography, experimental amyloidosis, screening
Introduction:
Crystallographic method involves the study of microcrystalline precipitation, representing the self-organized facies cluttering a biological fluid. Comparison of the structures of the composite model healthy blood enriched various metabolites (uric acid, proteins, creatinine, amino acids, cholesterol), the content data metabolites in the blood of patients (diabetes, obesity, gout, hypothyroidism, etc.) allowed to allocate generic microinclusions related metabolic disorders: hyperoxaemia, gierurikemii, hyperglycinemia, hypercalcemia, and hypercholesterolemia. Based on the results of these studies was set up a database of typical structures of the solid phase serum, which allowed to classify dehydrated drops on the biochemical composition. These methods of study of the morphological features of the solid body fluids are used with varying success in different areas of medicine.
1. Modeling of secondary amyloidosis
For modeling secondary amyloidosis, we used a biological model, which acted as a white mongrel male rats in the amount of 48 pieces. In this case, all the rats were divided into two groups: experimental and intact. All groups were strictly identical conditions vivarium AGMA and received an identical diet.The disease (secondary amyloidosis) was induced by administration of native ovalbumin in combination with Freund's complete adjuvant, which consisted of glycerol, gelatin and killed Mycobacterium tuberculosis. Experimental group received Freund's adjuvant and native egg albumin, and intact served as a control standard. Adjuvant in combination with native ovalbumin injected intraperitoneally for 7 days after day 1 ml per 300 g body weight.
On day 24 of the experiment, all animals were derived from it by drawing blood and internal organs for histological examination.
2. The method of Crystallography
The basis of this method has laid down wedge dehydration phenomenon, described in detail Martoussevitch. It consists in that the liquid droplet is placed on a rigid substrate passes certain phase dehydration to form a specific pattern crystallization. And it was the latter will be a substrate for the differential diagnosis of diseases.
For wedge dehydration method was taken 100 ul serum and 50 l of 0.9% solution NaCl, then the two solutions were mixed and the resulting substrate in a volume of 20 l was applied on a glass slide skimmed beforehand. Glass put in a petri dish for one hour at a temperature equal to 22oC and humidity equal to 55-75%. Thereafter, obtained by dehydration wedge facies analyzed under a microscope with a magnification of 100 times.
Focus is on the quality indicators, such as the form of crystals, their branching and spatial relationship. In the experimental group, we obtained large cruciform crystals which reduced its size
and closer to the thickened, dehydrated drops, the number of branches is minimized. In the same group intact, the crystals have characteristic "tender line", which differ significantly from those obtained in the first group of crystals. Also, a set of quantitative parameters are used as diagnostic criteria:
1. Number of nucleation sites in the two studies. Dimensions of the latter were obtained experimentally - we got the whole area of the drop, and then divided it into quadrants, with an area of 16 to 500 conventional units. In the experimental group, the number of nuclei is equal to 2 ± 1 (p = 0,05). In the same group of intact active component is 5 ± 1 (p = 0,05).
2. The number of secondary rays. In the group of animals with induced amyloidosis AL-active input parameter is 7 ± 2 (p = 0,05). The potential of the laboratory animal healthy intact group of secondary beams is 13 ± 2 (p = 0,05).
3. Protein banding pattern of the peripheral roll. Under experimental animals active parameter is 9 ± 2 (p = 0,05). It should be noted that these cracks are of the wrong type and direction. In the group of intact animals, the number of lines on the protein roller ravno12 ± 2 (p = 0,05), and the data lines are ordered strictly parallel course that also serves as the diagnostic criterion unlike facies AL-amyloidosis.
Results and discussion
As indicated above, the evaluation of the crystallogram can use the following parameters:
1. Quality:the form of crystals, their branching and spatial relationship.
2. Quantitative: number of nucleation sites, number of secondary rays and Protein banding pattern of the peripheral roll.
These options allow you to differentiate various pathologies at an experimental level. It is worth noting that in the future you can even use a picture of crystallization for the assessment of the severity of disease.All received and interpreted results of the analysis of the facies were compared with histological examination autoptatov organs (liver, kidney, spleen, lungs, heart) to confirm the correlation of the parameters and the presence of pathology. Based on this study, we obtained 100% confirmation of amyloidosis with histological results using the method developed. Materials and methods
To develop the method, we used the method of wedge dehydration droplet of which was proposed Martoussevitch. Secondary amyloidosis is reproduced on biological models. To confirm the presence of secondary amyloidosis Centre was performed histological staining of organs (kidney, liver, lung, spleen) Congo red using a polarizing microscope.
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Pic. 1. Painting crystallization rate
Pic. 2. Picture of crystallization in secondary amyloidosis
Conclusions
According to our research, we have developed a method for rapid diagnosis of secondary amyloidosis, which allows a short time with a high percentage of accuracy to determine the presence of disease.
References
1. Warsaw, VA, Proskurneva, EP The meaning and methods of morphological diagnosis of amyloidosis in modern medicine. Practical Nephrology. - 1998. - № 2. - P.16-23.
2. Houdner, P., Rausing, A., Steen, K., Torp A. Diagnosis of cardiac amyloidosis by Myocardial Biopsy. // Acta med. Scand - 1975-№6. p.525-528.
3. Skinner M. Amyloidosis Current Therapy in Allergy, Immunology, and Rheumatology. // Mosby-Year Book. - 1996. - p.235-240.
4. A. Vorobyov, Martusevich AK, SP Peretyagin Crystallogenesis biological fluids and substrates in assessing the state of the body. Nizhny Novgorod: FSE "NNIITO Medical Technologies", 2008. 384.
5.Martusevich AK Biokristallizatsiya: epistemology, methodology, data availability. Kirov: Typography Vyatka State Agricultural Academy, 2008. 15
Flow of pneumonia for children on the modern stage
YutkinaO.S., ArutyuanyanK.A.
Amur State Medical Academy, Blagoveshchensk, Russia Children's City Clinical Hospital, Blagoveshchensk, Russia
Abstracts: the analysis of the clinical course of the community-acquired pneumonia and estimation of adaptive reactions of organism of children.
Key words: children, community-acquired pneumonia.
In the structure of pulmonary pathology for children different ages community-acquired pneumonia make near 70-80%. Pneumonia tense included in a number 10 the most frequent reasons of death.Morbidity in the different regions of Russian averages from 4 to 17 cases pneumonia on 1000 child's population, having a necessity for hospitalizations, high lethality and social meaningfulness.At the analysis of frequency of pneumonia for the last 6 among child's population of the Amur area and Blagoveshchensk a tendency is educed to her height from 7 to 10 on the Amur area, from 4,2 to 7,5no Blagoveshchensk(approximately in 1,5 time).
At the detailed analysis evidently, that in 2009 frequency of pneumonias in Blagoveshchensk in 4 times exceeded previous 2008 year and 20,3h made further began to go down in 2010 - 14,5 in 2011 - 9,41 in 2012 - 7,5 this situation is related to the flash of Acute respiratory virus infection, in particular flu of AH1N1 (picture 1).
In connection with the complicated epidemiology situation in autumn of 2009 on the base of Children's City Clinical HospitalBlagoveshchensk hospitalization of children came true with suspicion on community-acquired pneumonia. In all for this period 564 children(in October - 194, November - 248, December - 122) were cured ((picture 2).
Materials and methods.
We are conduct a retrospective analysis 199 histories case of children with pneumonia, inpatient department of Children's City Clinical Hospital Blagoveshchensk in autumn of 2009.
We studied the adaptable reactions of organism of children were studied in the beginning of disease of pneumonia, at the moment of leaving the hospital and depending on age using the data of leucitogramm (percentage of limphocytes in leukocitar formula (L.H Garkavi, 1998).
Results and discussion.
Age-dependent structure of children: 1 month - 3th years old (11%) 4-6 years old (22%) in age 7-17 years old (67%),about from them 96% attended schools and kindergartens.
