RU2092839C1 - Method for differential diagnosis of toxemias and septicemia cases - Google Patents

Abstract

FIELD: medicine; diagnostics of infectious diseases. SUBSTANCE: patient's leucoconcentrate smear is analyzed by subjecting it to myeloperoxidase reaction. Purpose of analysis is to detect in smears characteristics forms of cells. In case of detecting in smears paraleucoblast forms, immature cells of myeloid series saturated with ferment-containing granules, and neutrophils having definitely marked polymorphism in terms of their size, diagnostician must recognize in third picture specific feature of septicemic sepsis. In presence in inflammation focus without septicemia, immature forms are usually associated with degranulated cells. EFFECT: method makes it possible to draw clear-cut boundary between detected septicemia and TOXEMIAS syndromes, to detect their combinations and to diagnose bacteriemia case during first 1-3 days after contracting disease. 2 cl, 3 dwg

Description

 The invention relates to medicine, namely to the diagnosis of infectious and inflammatory diseases.

 In clinical practice, the presence of toxemia syndrome is judged by a complex of subjective (headaches, dizziness, weakness, malaise, sweating), as well as objective (increased ESR, quantitative and qualitative changes in the leukocyte formula, as well as plasma proteins) symptoms. At the same time, it is known that these criteria are not strictly specific only for toxemia; certain combinations thereof also occur with septicemia.

 In order to diagnose septicemia, bacteriological methods are currently being used using a number of media: according to Hugh-Leifson; Gissa (for identification of staphylococcus), by Arons R.M. Kolker I.I. (for identification of Pseudomonas aeruginosa), etc.

 The bacteriological method using the Hugh-Leifson medium (V. S. Matkovsky et al. Infectious diseases. L. 1977, pp. 108-112) was taken as a prototype.

Composition of the medium: peptone 0.2% NaCl 0.5% K ₂ HPO ₄ 0.03% glucose 1% agar 0.3% bromothymol blue (indicator) 0.003% The prepared medium is poured into 5 ml tubes. Immediately before inoculation, tubes of medium are placed for 15 minutes in a boiling water bath to remove oxygen. The daily agar culture of the studied strain is sown in a loop in a column of medium by injection to the bottom of the tube, then the surface of the agar is poured with sterile liquid paraffin to create anaerobic conditions. Inoculation is incubated at 37 ^o C for 5 days, daily recording results. The reaction is regarded as positive if at least 2/3 of the medium is colored yellow.

 However, sterile blood culture methods are rarely used in clinics due to their technical complexity and length of time. It should also be noted that bacteriological analysis reveals the presence of microbes in the blood only in 10-18% of cases of sepsis (A.D. Ostrovsky, A.S. Vorobyov, 1985). This percentage increases slightly with repeated studies. And if we add to this the well-known fact that even with severe forms of sepsis, it is not always possible to isolate the pathogen due to the short duration of bacteremia, then a negative bacteriological test result should not be accepted as a sufficient reason to exclude the diagnosis of sepsis.

 The aim of the present invention is to increase the reliability of the diagnosis of toxemia and septicemia, as well as their combination in children.

The proposed method is based on the detection of smear of leukoconcentrate during the formulation of the response to myeloperoxidase characteristic cell forms:
in toxemia, a combination of degranulated neutrophils with immature forms (pri- and myelocytes saturated with peroxidase-containing granules) (PO granules).

 in septicemia, a combination of atypical blast cells (paralukoblasts) of the myeloid series with immature forms saturated with PO granules in the absence of degranulated neutrophils.

 with a combination of toxemia and septicemia, a combination of signs characteristic of the first two options, i.e. the presence in smears of degranulated cells, paraleukoblasts and immature forms of the neutrophilic series, excessively saturated with PO granules.

 Description of the proposed invention as a method.

 1. Obtaining leukoconcentrate. Heparinized venous blood in an amount of 3-5 ml settles for 20-30 minutes to precipitate red blood cells. The supernatant with suspended leukocytes is transferred to another tube and re-sedimented for 20-25 minutes. After removing the plasma from the precipitate, the thinnest smears are prepared. Shaking, centrifugation or the use of substances that accelerate the deposition of red blood cells is unacceptable.

2. The activity of myeloperoxidase in smears of the leukoconcentrate is detected by the improved benzidine method: thin smears from leukomass are dried for at least 40-60 minutes and fixed in freshly prepared alcohol-formol (1: 9). After washing with running water, the smears are incubated in a medium containing 2.5 ml of a solution of benzidine saturated in room temperature in Tris buffer (pH 7.2), 2.5 ml of distilled water and 1 ml of a 0.03% hydrogen peroxide solution for 5 min A saturated solution of benzidine in Tris buffer is prepared as follows: 90 mg of benzidine is added to 90 ml of Tris buffer heated to 70 ^° C and mixed thoroughly for 5-10 minutes. Then, after filtration through a cotton filter, the solution was allowed to cool to room temperature. At the same time, benzidine crystals settle to the bottom of the tube, which is an indicator of the saturation of the solution. We consider the use of such a solution to be essential both for standardizing the concentration of the substrate and for the subsequent obtaining of stable results. The solution is resistant for 2-3 months when stored in a dark place.

 The nuclei are stained with Ehrlich hematoxylin. For this, 10 drops of hematoxylin are added to 10 ml of distilled water, and after filtering through a paper filter, a few drops are applied for 2-3 minutes. At the same time, background staining of the cytoplasm is achieved, which is essential for the nature of the distribution of granules in the cells.

Examples of specific performance of the method:
1. A sick child A., 4 years old (IB No. 2694), was admitted to the somatic department of a multidisciplinary children's hospital with a diagnosis of acute pneumonia. Sick for two weeks, the disease began with cough, shortness of breath, high fever (38.8 ^o C).

 Objectively: the right half of the chest lags somewhat in the act of breathing. Percussion on the right below the angle of the scapula is blunted. Auscultatory, there are sonorous crepitating wheezing. Blood test: HB 72 units, CP 0.9, ESR 17 mm / h, L 4600.

 On the radiograph, the middle and lower lobes of the left lung are darkened. Clinical diagnosis: polysegmental pneumonia, moderate form. Sowing blood for sterility, carried out twice, the growth of microbes did not give.

When studying the described method revealed the following:
More than 60% of neutrophils have signs of degranulation, pronounced thinning of the granules, especially on the periphery of the cells, uneven location in the middle sections of the cytoplasm. In smears, single immature forms (po and myeloblasts) saturated with PO granules (Fig. 1). The picture is characteristic of toxemia. Recommendations are given.

2. Child Huseynov M. 6 months. (I.B. N 6350 (527)) was admitted to the thoracic department of the children's multidisciplinary hospital in Makhachkala. From 3 months of age, the child's temperature rose periodically to 38-39 ^o C. The umbilical wound did not heal for a long time. A month after the onset of the disease, vesicle-pustular rashes with liquid contents appeared on the skin. The condition of the child upon admission is severe, restless, capricious, and takes the breast poorly. Moderate cyanosis. In the lungs, vesicular breathing. The liver is increased by 3 cm. Hepatolienal syndrome.

Blood test: L 13.3 • 10 ⁹ , ESR 55 mm / hour. Preliminary diagnosis: staphylococcal sepsis? Sowing blood for sterility, carried out on the third day, the growth of microbes did not give.

 In the study using the described method, the following was revealed: most segmented neutrophils have high and moderate activity, there are no signs of their degranulation. On smears, not a few (2%) of atypical blast cells-paraleukoblasts slightly segmented by the nucleus and traces of diffuse or granular activity (figure 2). These forms alternate with immature cells of the myeloid series (promyelocytes and myelocytes), saturated with peroxidase-containing granules. Noticeable polymorphism of neutrophils in size. The picture is characteristic of septicemia.

 Recommendations are given. At repeated (in 10 days) bacteriological analysis is sown.

3. A 7-year-old patient (i.p. N 4449) was admitted to the surgical department of a children's multidisciplinary hospital with a diagnosis of destructive pneumonia. The general condition at admission is severe, groans, severe shortness of breath, temperature - 38 ^o C.

 Shortening percussion sound in the lungs on the right. Auscultatory breathing with a bronchial tinge.

 X-ray of the chest: to the right of the VIII rib to the diaphragm intense uniform dimming of the pulmonary field with a horizontal mark.

Blood test: ESR 66 mm / h, L 14.5 • 10 ⁹ , myel 7, 10, n -13, s 42.

 Sowing blood for sterility gave rise.

 Clinical diagnosis: acute bacterial destruction of the right lung. Sepsis.

 In the study using the described method, it was revealed that neutrophils with signs of degranulation (45%) alternate with immature cells (pro and myelocytes), saturated PO granules (3%), and paraleukoblastic forms of small sizes with weak diffuse enzymatic activity in the cytoplasm (4%) ( Fig. 3).

 The picture is characteristic of toxemia and septicemia.

 Repeated bacteriological analysis is also seeded.

In order to verify the reliability of the results obtained on clinical material, experiments were performed with the reproduction of septicemia and toxemia. Sepsis in rats was obtained by the method of L. L. Shimkevich by a single intramuscular injection of a daily culture containing 1 • 10 ⁵ microbial bodies in 1 ml. In all animals, bacteriological analysis was seeded in the first 1-3 days.

 After staging the reaction to myeloperoxidase, the following smears were found in smears of leukoconcentrate: paraleukoblastic forms (1%) with sharp size polymorphism and traces of granular activity, single immature forms (promyelocytes) saturated with PO granules. Mature forms of neutrophils without signs of degranulation.

With the introduction of microbial bodies in a 10% CaCl ₂ solution, which usually causes local tissue necrosis, a large percentage (55-65%) of degranulated segmented neutrophils are found in smears along with paralukoblasts and immature forms of the myeloid series.

Distinctive features of the prototype:
set the reaction to myeloperoxidase (and according to the prototype, the strain is inoculated into a medium column and incubated at 37 ^° C for 5 days);
when degranulated neutrophils appear in smears in combination with immature forms (pro- and myelocytes), saturated with PO granules, diagnose toxemia;
when atypical blast forms (paraleukoblasts) appear in smears with a sharp polymorphism of sizes, as well as nuclear forms in combination with immature myeloid cells and neutrophils without signs of degranulation, sepsis is diagnosed;
with the appearance in the smears of degranulated cells of paraleukoblasts and immature forms of the neutrophilic series, excessively saturated with PO granules, toxemia and septicemia are diagnosed;
the solution is prepared as follows: 2.5 ml of distilled water and 1 ml of a 0.03% hydrogen peroxide solution are added to 2.5 ml of a solution of benzidine saturated in room temperature in Tris buffer (pH 7.2). To obtain a saturated solution of benzidine, 90 mg of benzidine are added to 90 ml of Tris buffer heated to 70 ^° C and mixed thoroughly. Then, after filtration through a cotton filter, the solution was allowed to cool to room temperature. At the same time, benzidine crystals settle to the bottom of the tube, which is an indicator of the saturation of the solution.

Claims (2)

 1. A differential method for the diagnosis of toxemia and septicemia, including a study of the patient’s blood, characterized in that smears of the leukoconcentrate are prepared from the patient’s blood and a reaction is made for myeloperoxidase and in the presence of degranulated neutrophils in the smears (50–70%) in combination with immature forms (pro- and myelocytes), saturated with peroxidase-containing granules (2 - 4%), diagnose toxemia in the presence of atypical blast forms (parleycloblast) with a sharp polymorphism of sizes, as well as nuclear shapes (3 5%) in combination with immature cells oidnogo of (pro- and myelocytes) in an amount of 1 3% neutrophils and degranulation without any signs of septicemia is diagnosed, and in the presence of degranulated neutrophils and immature forms paraleykoblastov myeloid series, saturated peroksidazosoderzhaschimi granules diagnose septicemia and toxemia combination. 2. The method according to claim 1, characterized in that the solution for staging the reaction to myeloperoxidase is prepared at room temperature, pouring 2.5 ml of distilled water and 1 to 2.5 ml of a saturated solution of benzidine in Tris buffer (pH 7.2) and ml of a 0.03% hydrogen peroxide solution, while a saturated solution of benzidine is obtained by mixing 90 ml of Tris buffer heated to 70 ^° C with 90 mg of benzidine and then filtering it through a cotton swab.

Images