MD1014Z - Method for differential diagnosis of endogenous intoxication caused by b-hemolytic streptococcus or tuberculosis mycobacterium in different age groups - Google Patents
Method for differential diagnosis of endogenous intoxication caused by b-hemolytic streptococcus or tuberculosis mycobacterium in different age groups Download PDFInfo
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- MD1014Z MD1014Z MDS20150071A MDS20150071A MD1014Z MD 1014 Z MD1014 Z MD 1014Z MD S20150071 A MDS20150071 A MD S20150071A MD S20150071 A MDS20150071 A MD S20150071A MD 1014 Z MD1014 Z MD 1014Z
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- endogenous intoxication
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Abstract
Description
Invenţia se referă la medicină, în special la imunologie şi poate fi utilizată pentru diagnosticul diferenţiat al intoxicaţiei endogene cauzate de streptococul β-hemolitic sau de micobacteria tuberculozei la diferite grupuri de vârstă. The invention relates to medicine, in particular to immunology and can be used for the differential diagnosis of endogenous intoxication caused by β-hemolytic streptococcus or mycobacterium tuberculosis in different age groups.
În calitate de cea mai apropiată soluţie este cunoscută metoda de determinare a intoxicaţiei endogene, care constă în determinarea indicelui leucocitar al intoxicaţiei (ILI), propusă de [Kalf-Khalif, 1941]. Metoda este bazată pe cuantificarea diferitor grupări celulare din şirul leucocitar sangvin, iar ILI se determină în baza relaţiei: As the closest solution, the method of determining endogenous intoxication is known, which consists in determining the leukocyte intoxication index (ILI), proposed by [Kalf-Khalif, 1941]. The method is based on the quantification of different cell groups in the blood leukocyte series, and the ILI is determined based on the relationship:
, ,
unde: MIE - mielocite, T - tinere, N - neutrofile nesegmentate, S - neutrofile segmentate, CP - celule plasmatice, М - monocite, L - limfocite, E - eozinofile. where: MIE - myelocytes, T - young, N - unsegmented neutrophils, S - segmented neutrophils, CP - plasma cells, М - monocytes, L - lymphocytes, E - eosinophils.
În normă acest indice constituie 0,3…1,5 unităţi. Normally, this index is 0.3…1.5 units.
În procesele inflamatorii, indicele leucocitar al intoxicaţiei este crescut. Creşterea nivelului ILI până la 4…9 indică asupra componentei bacteriene ponderabile a intoxicaţiei endogene. Creşterea ILI este determinată de dispariţia eozinofilelor, mărirea conţinutului de componente leucocitare segmentate, celule plasmatice şi micşorarea numărului de limfocite. Indicele leucocitar al intoxicaţiei Kalf-Khalif ia în calcul un şir de caracteristici bazate pe apartenenţa funcţională a diferitor grupuri celulare incluse în formula leucocitară. ILI este un indice simplu, accesibil şi destul de informativ cu privire la gravitatea intoxicaţiei endogene, utilizat în practica clinică. In inflammatory processes, the leukocyte intoxication index is increased. An increase in the ILI level to 4…9 indicates a significant bacterial component of endogenous intoxication. The increase in ILI is determined by the disappearance of eosinophils, an increase in the content of segmented leukocyte components, plasma cells and a decrease in the number of lymphocytes. The Kalf-Khalif leukocyte intoxication index takes into account a number of characteristics based on the functional affiliation of various cell groups included in the leukocyte formula. ILI is a simple, accessible and quite informative index of the severity of endogenous intoxication, used in clinical practice.
Dezavantajul metodei descrise constă în aceea că exactitatea ei scade semnificativ odată cu progresarea intoxicaţiei endogene care este însoţită de suprimarea mecanismelor de protecţie imună şi formarea semnelor de insuficienţă organică. De asemenea, drept neajuns al metodei poate fi considerat şi faptul că odată cu apariţia la bolnavi a semnelor de alergizare (conţinut înalt de eozinofile), formula de calcul al ILI nu funcţionează, deci nu poate fi aplicată, totodată nu se determină specificul intoxicaţiei endogene şi nu poate fi făcut un diagnostic diferenţiat. The disadvantage of the described method is that its accuracy decreases significantly with the progression of endogenous intoxication, which is accompanied by the suppression of immune protection mechanisms and the formation of signs of organic insufficiency. Also, the fact that with the appearance of signs of allergy in patients (high eosinophil content), the ILI calculation formula does not work, therefore it cannot be applied, at the same time the specificity of endogenous intoxication is not determined and a differential diagnosis cannot be made can be considered a shortcoming of the method.
Problema pe care o rezolvă invenţia propusă constă în elaborarea unei metode de determinare a specificităţii intoxicaţiei endogene care ar fi eficientă la pacienţii cu niveluri înalte ale eozinofilelor la diferite grupuri de vârstă şi ar putea fi utilizată pe larg în practica de laborator. The problem solved by the proposed invention consists in developing a method for determining the specificity of endogenous intoxication that would be effective in patients with high eosinophil levels in different age groups and could be widely used in laboratory practice.
Metoda, conform invenţiei, înlătură dezavantajele menţionate mai sus prin aceea că se prepară o suspensie de leucocite prelevate de la pacient, se determină cantitatea finală a biomarkerului TNF-α conform formulei: The method, according to the invention, eliminates the disadvantages mentioned above by preparing a suspension of leukocytes taken from the patient, determining the final amount of the TNF-α biomarker according to the formula:
x10000, x10000,
în cazul în care la copii de până la 18 ani pentru TNF-α final se determină valori cuprinse în intervalul 2,0…2,07 se diagnostichează o intoxicaţie endogenă cauzată de streptococul β-hemolitic, iar în cazul în care valorile sunt mai mari de 2,08 se diagnostichează o intoxicaţie endogenă cauzată de micobacteria tuberculozei, în cazul când la maturi de peste 18 ani pentru TNF-α final se determină valori cuprinse în intervalul 6,55…6,87 se diagnostichează o intoxicaţie endogenă cauzată de streptococul β-hemolitic, iar când se obţin valori mai mari de 6,88 se diagnostichează o intoxicaţie endogenă cauzată de micobacteria tuberculozei. If in children up to 18 years of age, final TNF-α values within the range of 2.0…2.07 are determined, endogenous intoxication caused by β-hemolytic streptococcus is diagnosed, and if the values are higher than 2.08, endogenous intoxication caused by mycobacterium tuberculosis is diagnosed, if in adults over 18 years of age, final TNF-α values within the range of 6.55…6.87 are determined, endogenous intoxication caused by β-hemolytic streptococcus is diagnosed, and when values higher than 6.88 are obtained, endogenous intoxication caused by mycobacterium tuberculosis is diagnosed.
Rezultatul invenţiei constă în crearea unei metode de determinare a specificităţii intoxicaţiei endogene eficiente la pacienţii cu niveluri înalte ale eozinofilelor la diferite grupuri de vârstă care ar putea fi utilizată pe larg în practica de laborator. The result of the invention consists in creating a method for determining the specificity of endogenous intoxication effective in patients with high eosinophil levels in different age groups that could be widely used in laboratory practice.
Intoxicaţia endogenă este un sindrom polietiologic şi polipatogen caracterizat prin acumularea în ţesuturi şi lichidele biologice a substanţelor endogene toxice. Endogenous intoxication is a polyetiological and polypathogenic syndrome characterized by the accumulation of toxic endogenous substances in tissues and biological fluids.
Printre sursele de endotoxine pot fi nominalizate: Among the sources of endotoxins can be mentioned:
- produsele dezintegrării celulare, improprii sistemului imunitar, antigenii şi complexele agresoare imune; - cellular disintegration products, inappropriate for the immune system, antigens and immune aggressor complexes;
- toxinele microbiene (exo- şi endotoxinele) şi alţi factori ai patogenităţii microorganismelor (patogene, condiţionat patogene, nepatogene); - microbial toxins (exo- and endotoxins) and other factors of the pathogenicity of microorganisms (pathogenic, conditionally pathogenic, non-pathogenic);
- mediatorii inflamaţiei, citokinele, prostaglandinele, leucotrienele, proteinele fazei acute ş.a. compuşi biologic activi. - inflammatory mediators, cytokines, prostaglandins, leukotrienes, acute phase proteins, etc. biologically active compounds.
Unul dintre mediatorii endogeni de bază ai inflamaţiei şi posibil unul dintre agenţii universali implicaţi în realizarea sindromului intoxicaţiei endogene este factorul de necroză tumorală, exprimat prin cele două forme ale sale - TNF-α şi TNF-β. Este capabil să inducă in vivo necroza hemoragică a unor celule tumorale, fără a afecta celulele normale. Pe lângă aceasta, induce şocul dacă elaborarea lui este condiţionată de endotoxinele bacteriene. One of the basic endogenous mediators of inflammation and possibly one of the universal agents involved in the development of the endogenous intoxication syndrome is tumor necrosis factor, expressed in its two forms - TNF-α and TNF-β. It is capable of inducing hemorrhagic necrosis of some tumor cells in vivo, without affecting normal cells. In addition, it induces shock if its development is conditioned by bacterial endotoxins.
În condiţii normale factorul de necroză tumorală este elaborat de către multe tipuri de celule (fagocitele mononucleare, limfocitele B, limfocitele T, celulele NK (natural killer), leucocitele polimorfonucleare, mastocite şi bazofile, celulele endoteliului vaselor). Under normal conditions, tumor necrosis factor is produced by many types of cells (mononuclear phagocytes, B lymphocytes, T lymphocytes, NK (natural killer) cells, polymorphonuclear leukocytes, mast cells and basophils, vascular endothelial cells).
Titrurile sale cresc la infectare, când în organism pătrund endotoxinele bacteriene. Concentraţiile înalte, determinate în caz de sepsis gram-negativ, reprezintă cauza principală a instalării şocului septic. Its titers increase during infection, when bacterial endotoxins enter the body. High concentrations, determined in case of gram-negative sepsis, are the main cause of septic shock.
Din aceste considerente, TNF-α a fost selectat în calitate de marker biologic al intoxicaţiei endogene. For these reasons, TNF-α was selected as a biological marker of endogenous intoxication.
Avantajele metodei revendicate constau în: The advantages of the claimed method consist of:
- determinarea specificităţii intoxicaţiei endogene, care este imposibilă în cazul aplicării celei mai apropiate metode; - determining the specificity of endogenous intoxication, which is impossible when applying the closest method;
- aplicarea metodei revendicate permite a efectua analiza diferenţiată între etiologia streptococică şi cea tuberculoasă a intoxicaţiei endogene; - the application of the claimed method allows to perform the differentiated analysis between the streptococcal and tuberculous etiology of endogenous intoxication;
- metoda propusă a identificat intoxicaţia endogenă specifică de 22 ori mai frecvent la copiii cu tuberculoză şi de 2 ori mai frecvent la maturii cu tuberculoză la antigenii micobacteriei tuberculoase, comparativ cu aplicarea indicelui leucocitar al intoxicaţiei Kalf-Khalif; - the proposed method identified specific endogenous intoxication 22 times more frequently in children with tuberculosis and 2 times more frequently in adults with tuberculosis to mycobacterium tuberculosis antigens, compared to the application of the Kalf-Khalif leukocyte intoxication index;
- exprimarea producţiei induse de TNF-α în pg/ml/10000 celule permite obţinerea unor rezultate precise indiferent de cantitatea de celule izolate la un bolnav sau altul, sau de pierderile de celule la procedura de spălare. - expressing the induced TNF-α production in pg/ml/10,000 cells allows obtaining precise results regardless of the quantity of cells isolated from one patient or another, or the loss of cells during the washing procedure.
Exemplu concret de realizare a invenţiei Concrete example of realization of the invention
Metoda revendicată constă din următoarele etape: izolarea celulelor, cultivarea şi determinarea cantităţii biomarkerului TNF-α. The claimed method consists of the following steps: cell isolation, cultivation and determination of the amount of TNF-α biomarker.
Etapa 1. Într-o eprubetă sterilă se prelevă sânge din vena cubitală în volum de 3,0 ml. În prealabil, în eprubetă se toarnă soluţie de heparină pe mediul 199, reieşind din calculul 25 UN la 1,0 ml sânge. Se prepară amestecul pentru sedimentarea sângelui (3 părţi mediu 199 şi 1 parte gelatină 10%) în volum de 3,0 ml. Sângele şi amestecul pentru sedimentare se amestecă şi se plasează într-un termostat pentru 30 min la 37°С în poziţie verticală pentru sedimentarea eritrocitelor. După care, suprenatantul ce conţine suspensia celulară leucocitară se colectează într-o eprubetă curată şi se centrifughează timp de 10 min la o forţă relativă de centrifugare (RCF) de 175 RCF. După aceasta supernatantul se aruncă, iar celulele rămase se resuspendă în 3,0 ml mediu 199. Într-o eprubetă curată se toarnă câte 1,0 ml suspensie celulară şi se adaugă câte 2,0 ml mediu 199. În eprubetă se numără celulele după metoda standard utilizând camera Goreaev. În acest timp are loc o producere spontană de TNF-α. Step 1. In a sterile test tube, blood is taken from the cubital vein in a volume of 3.0 ml. First, a heparin solution is poured into the test tube on medium 199, based on the calculation of 25 UN per 1.0 ml of blood. The blood sedimentation mixture is prepared (3 parts medium 199 and 1 part gelatin 10%) in a volume of 3.0 ml. The blood and sedimentation mixture are mixed and placed in a thermostat for 30 min at 37°С in a vertical position for erythrocyte sedimentation. After that, the supernatant containing the leukocyte cell suspension is collected in a clean test tube and centrifuged for 10 min at a relative centrifugal force (RCF) of 175 RCF. After this, the supernatant is discarded and the remaining cells are resuspended in 3.0 ml of medium 199. Pour 1.0 ml of cell suspension into a clean test tube and add 2.0 ml of medium 199. Count the cells in the test tube according to the standard method using the Goreaev chamber. During this time, a spontaneous production of TNF-α occurs.
Etapa 2. Cultura obţinută în etapa 1 se plasează în termostat la o temperatură de 35°…37°С şi se incubează timp de 48 ore. Stage 2. The culture obtained in stage 1 is placed in a thermostat at a temperature of 35°…37°С and incubated for 48 hours.
Etapa 3. Cultura de celule se resuspendă în mediul pentru creştere şi se centrifughează timp de 10 min la 175 RCF. După care, se prelevă supernatantul şi prin metoda de analiză imunoenzimatică standard se determină titrul TNF-α. Pentru determinarea TNF-α în metoda propusă s-a utilizat kit-ul de reactivi ai firmei "Вектор-БЕСТ" (Federaţia Rusă). Step 3. The cell culture is resuspended in the growth medium and centrifuged for 10 min at 175 RCF. After that, the supernatant is taken and the TNF-α titer is determined by the standard immunoenzymatic analysis method. For the determination of TNF-α in the proposed method, the reagent kit of the company "Vector-BEST" (Russian Federation) was used.
Dat fiind că la fiecare dintre bolnavi se izolează o cantitate diferită de celule producătoare de TNF-α, pentru alinierea datelor obţinute rezultatul final al producţiei de TNF-α final se calculează în baza formulei: Given that a different amount of TNF-α producing cells is isolated from each patient, to align the data obtained, the final result of TNF-α production is calculated based on the formula:
x10000, x10000,
şi se exprimă în pg/ml/10000 celule. and is expressed in pg/ml/10000 cells.
Exemplul 1 Example 1
Bolnavul A., 4 ani, diagnosticul - tonsilită cronică decompensată. Patient A., 4 years old, diagnosis - chronic decompensated tonsillitis.
În eprubeta după incubare s-a determinat cantitatea TNF-α care a constituit 9,9 pg/ml, iar cantitatea totală a celulelor în eprubetă a constituit 48231 unităţi, respectiv rezultatul final al producţiei de TNF-α calculat este: In the test tube after incubation, the amount of TNF-α was determined to be 9.9 pg/ml, and the total amount of cells in the test tube was 48231 units, respectively the final result of the calculated TNF-α production is:
În cazul dat se diagnostichează o intoxicaţie endogenă cauzată de streptococul β-hemolitic. In this case, an endogenous intoxication caused by β-hemolytic streptococcus is diagnosed.
Exemplul 2 Example 2
Bolnavul B., 7 ani, diagnosticul - tonsilită cronică compensată. Patient B., 7 years old, diagnosis - compensated chronic tonsillitis.
În eprubeta după incubare s-a determinat cantitatea TNF-α care a constituit 15,6 pg/ml, iar cantitatea totală a celulelor în eprubetă a constituit 56486 unităţi, respectiv rezultatul final al producţiei de TNF-α calculat este: In the test tube after incubation, the amount of TNF-α was determined to be 15.6 pg/ml, and the total amount of cells in the test tube was 56486 units, respectively the final result of the calculated TNF-α production is:
În cazul dat se diagnostichează o intoxicaţie endogenă cauzată de micobacteria tuberculozei. In this case, an endogenous intoxication caused by mycobacterium tuberculosis is diagnosed.
Exemplul 3 Example 3
Bolnavul B., 19 ani, diagnosticul - tonsilită cronică compensată. Patient B., 19 years old, diagnosis - compensated chronic tonsillitis.
În eprubeta după incubare s-a determinat cantitatea TNF-α care a constituit 39,2 pg/ml, iar cantitatea totală a celulelor în eprubetă a constituit 54682 unităţi, respectiv rezultatul final al producţiei de TNF-α calculat este: In the test tube after incubation, the amount of TNF-α was determined to be 39.2 pg/ml, and the total amount of cells in the test tube was 54682 units, respectively the final result of the calculated TNF-α production is:
În cazul dat se diagnostichează o intoxicaţie endogenă cauzată de micobacteria tuberculozei. In this case, an endogenous intoxication caused by mycobacterium tuberculosis is diagnosed.
1. Кальф-Калиф Я.Я. О лейкоцитарном индексе интоксикации и его практическом значении. Врачебное дело, 1941, N. 1, c. 31-35 1. Кальф-Калиф Я.Я. About the leukocytic index of intoxication and its practical significance. Medical case, 1941, N. 1, c. 31-35
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| MD963G2 (en) * | 1997-06-20 | 1999-05-31 | Государственный Медицинский И Фармацевтический Университет "Nicolae Testemitanu" Республики Молдова | Floss |
| RU2193780C1 (en) * | 2001-04-26 | 2002-11-27 | Саратовский научно-исследовательский институт травматологии и ортопедии | Method for determining endogenous intoxication |
| UA52475A (en) * | 2002-06-12 | 2002-12-16 | Інститут Хірургії Та Трансплантології Академії Медичних Наук України | Method for diagnosing endogenous intoxication |
| MD20010417A (en) * | 2001-12-19 | 2003-09-30 | Univ Nicolae Testemitanu | Antirecidivating method of treatment of rhinosinous polyposis associated with bronchial asthma |
| MD3770F1 (en) * | 2008-08-22 | 2008-12-31 | Centrul National Stiintifico-Practic De Medicina Preventiva | Method for noninfluenzal acute viral respiratory infection prophylaxis |
| UA46403U (en) * | 2009-05-25 | 2009-12-25 | Андрей Анатольевич Воронко | METHOD FOR DIAGNOSTICS OF SEVERITY OF endogenous INTOXICATION AT METABOLIC SYNDROME |
| RU2395087C2 (en) * | 2008-06-23 | 2010-07-20 | Российская Федерация, от имени которой выступает Федеральное государственное образовательное учреждение высшего профессионального образования "Саровский государственный физико-технический институт" (ФГОУ ВПО"СарФТИ") | Method of diagnosis of endogenous intoxication |
| UA53882U (en) * | 2010-03-26 | 2010-10-25 | Винницкий Национальный Медицинский Университет Им. Н.И. Пирогова | Method for determination of endogenous intoxication of organism at disorder of large intestine transit |
| UA61220U (en) * | 2010-12-29 | 2011-07-11 | Украинский Научно-Исследовательский Институт Медицинской Реабилитации И Курортологии | Method for determination of endogenous intoxication |
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2015
- 2015-05-28 MD MDS20150071A patent/MD1014Z/en not_active IP Right Cessation
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| RU2122739C1 (en) * | 1996-02-02 | 1998-11-27 | Миронов Петр Иванович | Method of diagnosing gravity of endogenous intoxication in cases of acute purulo-septic diseases in children |
| MD667F1 (en) * | 1996-02-05 | 1997-01-31 | Asociatia De Productie Aroma | Rachiu (variant) Vodka (variant) |
| MD805F1 (en) * | 1997-03-31 | 1997-08-31 | Inst Genetica Rep Moldova | Composition for presowing maize seeds treetment |
| MD963G2 (en) * | 1997-06-20 | 1999-05-31 | Государственный Медицинский И Фармацевтический Университет "Nicolae Testemitanu" Республики Молдова | Floss |
| RU2193780C1 (en) * | 2001-04-26 | 2002-11-27 | Саратовский научно-исследовательский институт травматологии и ортопедии | Method for determining endogenous intoxication |
| MD20010417A (en) * | 2001-12-19 | 2003-09-30 | Univ Nicolae Testemitanu | Antirecidivating method of treatment of rhinosinous polyposis associated with bronchial asthma |
| UA52475A (en) * | 2002-06-12 | 2002-12-16 | Інститут Хірургії Та Трансплантології Академії Медичних Наук України | Method for diagnosing endogenous intoxication |
| RU2395087C2 (en) * | 2008-06-23 | 2010-07-20 | Российская Федерация, от имени которой выступает Федеральное государственное образовательное учреждение высшего профессионального образования "Саровский государственный физико-технический институт" (ФГОУ ВПО"СарФТИ") | Method of diagnosis of endogenous intoxication |
| MD3770F1 (en) * | 2008-08-22 | 2008-12-31 | Centrul National Stiintifico-Practic De Medicina Preventiva | Method for noninfluenzal acute viral respiratory infection prophylaxis |
| UA46403U (en) * | 2009-05-25 | 2009-12-25 | Андрей Анатольевич Воронко | METHOD FOR DIAGNOSTICS OF SEVERITY OF endogenous INTOXICATION AT METABOLIC SYNDROME |
| UA53882U (en) * | 2010-03-26 | 2010-10-25 | Винницкий Национальный Медицинский Университет Им. Н.И. Пирогова | Method for determination of endogenous intoxication of organism at disorder of large intestine transit |
| UA61220U (en) * | 2010-12-29 | 2011-07-11 | Украинский Научно-Исследовательский Институт Медицинской Реабилитации И Курортологии | Method for determination of endogenous intoxication |
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| MD1014Y (en) | 2016-03-31 |
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