RU2298183C2 - Method for detecting myeloperoxidase activity in sputum neutrophils and eosinophils - Google Patents

Abstract

FIELD: medicine, diagnostics.

SUBSTANCE: one should study fresh sputum smears (1-d-long storage, not more) which are to be fixed with formalin-alcohol solution, washed in running water, dried, poured as a reagent onto peroxidase, washed out with distilled water, permeated with a filter paper and finished dyeing with azure-1. Then it is necessary to photograph them in a light microscope at x1000 magnification, at maximal resolution, a scanogram should be imported into Photoshop CS program to detect the intensity of dyeing of cytochemical reaction products in blue spectrum, express it in reverse units of density and at intensity value being equal to 107.2±2.5 reverse units of density one should detect the norm for neutrophils and at 59.2±4.3 reverse units of density - for eosinophils, at values exceeding the values mentioned one should determine low activity of myeloperoxidase in cells, at values being under the mentioned values one should determine high activity of myeloperoxidase. The innovation enables to increase information value in detecting myeloperoxidase activity in sputum neutrophils and eosinophils.

EFFECT: higher accuracy of detection.

1 dwg

Description

The invention relates to medicine, diagnostics, and can be used to determine the activity of myeloperoxidase in neutrophilia and eosinophils of sputum.

A known method for the quantitative determination of myeloperoxidase activity in fluid samples of bronchoalveolar lavage in patients with cystic fibrosis, proposed by K. Paul, E. Rietschel et al. (Amer. Jour. Of Resp. And Crit Care, vol. 169, 2004). The reaction is monitored at a wavelength of 470 nm using an Arospec III apparatus, Germany. Myeloperoxidase activity is defined as the correspondence of 1 unit of myeloperoxidase activity to the consumption of 1 mmol of hydrogen peroxide. However, the known method is not sufficiently accurate and informative, since it does not allow to quantify the activity of myeloperoxidase in a (individual) specific cell.

A new technical problem is to increase the accuracy and information content of the method.

The problem is solved by a new method for determining the activity of myeloperoxidase in sputum neutrophils and eosinophils, consisting in the study of sputum, whereby freshly prepared sputum smears are examined, for which a light microscope with a × 1000 digital camera Sony DSC-S70 photographs eosinophils and neutrophils with maximum resolution, photographs they are imported into Adobe Photoshop, after which, using the Photoshop CS software package, the following actions are performed: using the lasso function, a neutrophil is extracted whether eosinophil, in the histogram function, the cell area in pixels is read, the magic wand function removes the nucleus from the cell, then the intensity of the color of the products of the cytochemical reaction in the blue spectrum is read in the histogram function, the intensity is expressed in inverse units of density and with an intensity value equal to 107.2 ± 2.5 arr. units square, for neutrophils and 59.2 ± 4.3 arr. unit raft. for eosinophils, the norm is determined at indices that exceed the indicated intensities; low myeloperoxidase activity in cells is determined; at indices below the indicated intensities, high myeloperoxidase activity is determined.

The method is as follows.

Freshly prepared smears of no more than one day ago are fixed in an alcohol-formalin mixture for 30 seconds.

Washed in running water, dried.

Glasses with sputum smears are placed on the rails and filled with a myeloperoxidase reagent (10 parts of benzidine, 60 ml of 96% ethanol, 40 ml of distilled water, 0.2 ml of a 3% hydrogen peroxide solution) for 20 minutes.

Glasses with sputum smears are washed with distilled water for 10 seconds and blotted with filter paper.

Smears are stained with azure-1 dye for 10-15 seconds.

Glasses are washed in running water.

Then, using a light microscope at a magnification of × 1000, the Sony DSC-S70 digital camera, with the maximum resolution, takes photos of eosinophils and neutrophils, imports the scenogram into Adobe Photoshop, and then performs the following actions using the Photoshop CS software package: using the lasso function neutrophil or eosinophil is isolated, the cell area indicator in pixels is read in the histogram function, the nucleus is removed from the cell with the magic wand function, then the color intensity of the product is read in the histogram function comrade cytochemical reaction in the blue spectrum, the feedback sensitivity of 10 to 60 depending on the degree of image sharpness intensity is expressed in units of inverse density (sp. U. pl.) and at a value of intensity equal to 107.2 ± 2.5 mod. units square, for neutrophils and 59.2 ± 4.3 arr. unit raft. for eosinophils, the norm is determined, at indicators exceeding the indicated intensity values, low myeloperoxidase activity in the cells is determined, at indicators below the indicated intensity values, high myeloperoxidase activity is determined.

The proposed criteria are selected based on the interpretation of the results of experimental studies of sputum samples in a total of 50 patients with chronic obstructive pulmonary disease and 25 healthy non-smokers, control groups whose color intensity values of the products of the cytochemical reaction in the control group were 107.2 + -2.5 arr. units square, for neutrophils and 59.2 + -4.3 arr. unit raft. for eosinophils, respectively. Indicators exceeding the indicated intensities indicated a low activity of myeloperoxidase in the cells, indicators below the indicated intensities indicated a high enzyme activity.

The average color intensity of the products of the cytochemical reaction in patients with COPD was 96.5 ± 1.4 and 55.8 ± 2.9 arr. units pl. for neutrophils and eosinophils, respectively, indicating a high enzyme activity in induced sputum cells in patients compared with the control group.

The advantage of the proposed method is high accuracy, the lack of subjective perception by the researcher of the color and brightness of the color of the drug, the ability to use a nominal scale. This makes it possible to increase the accuracy and informativeness of determining the activity of myeloperoxidase in induced sputum cells, which seems important in the diagnosis and in terms of further research on the problem of chronic obstructive pulmonary disease.

Claims (1)

A method for determining the activity of myeloperoxidase in sputum neutrophils and eosinophils, including the study of cells in smears of a biological sample, which are fixed with formalin-alcohol solution, washed in running water, dried, filled with a peroxidase reagent containing 96% benzidine, ethyl alcohol, distilled water, hydrogen peroxide, stained with dye, washed in running water and dried, characterized in that they examine freshly prepared sputum smears no more than one day old, staining is performed for 20 min, washed with distilled water for 10 s, blotted with filter paper, stained with Azur-1 dye for 10-15 s, then photographed in a light microscope at a magnification of × 1000, with a maximum resolution, the scan is imported into Photoshop CS, determine the color intensity of the products of the cytochemical reaction in the blue spectrum, express it in inverse units of density and with a magnitude of intensity equal to 107.2 ± 2.5 arr. units square, determine the norm for neutrophils and 59.2 ± 4.3 arr. units pl. for eosinophils, with indicators exceeding the specified values, low myeloperoxidase activity in the cells is determined, with indicators below the specified values, high myeloperoxidase activity is determined.