Method for immunohistochemical staining of cryostat tissue sections under conditions of intraoperative diagnosis

FIELD: medicine.

SUBSTANCE: tissue sections are incubated simultaneously with mixed primary and secondary antibodies. Primarily, the primary antibodies are applied on the tissues, and in 15 seconds of the incubation process - the secondary antibodies without washing with a buffer. It is followed with combined incubation for 4 minutes. A substrate-chromogen solution for the incubation is prepared immediately before the application, the sections are washed with a buffer solution and distilled water heated to temperature 37°C.

EFFECT: method allows fast and precise tumour histogenesis, detection of tumour cells within resection boundaries.

6 dwg, 2 tbl, 6 ex

 

The invention relates to medicine, namely to immunohistochemical studies, and can be used for immunophenotyping of tumors in terms of intraoperative diagnosis.

Currently immunohistochemical research methods are an integral part in the diagnosis of tumors. In the study of tissues immunohistochemical conclusion, on the one hand, confirms the morphological diagnosis, on the other hand, when unclear morphological picture helps the pathologist to determine the histogenesis of the tumor. Immunohistochemistry tissues - a method of identifying antigenic structures of tissues. This is one of the most modern methods of differential diagnostics of oncological diseases, allowing to determine the histogenesis of the tumor at the molecular level.

The loss of the morphological features of cells in different types of tumors, especially when metastasis and undifferentiated neoplasms small cell and polymorphic cellular nature, does not allow for differential diagnosis of tumors using only morphological methods. The most important method of clarifying diagnostic immunohistochemical. It allows you to define immunophenotype tumor, to investigate its biological properties, to determine the molecular-biological prognostic factors.

In some PA the ideological States, especially tumors, it is difficult and even impossible using histo - or cytological stains to determine the type of tissue, its origin. Similar difficulties arise when determining the type of pathogen infection. Meanwhile, the precise verification process is of great importance for diagnosis, treatment and prognosis of the disease. Therefore, it is advisable to use a variety of additional methods of research. One of these is the immunohistochemical method, the principle of which is that on histo - or cytological preparations applied solutions with antibodies to the target antigen is a tumor, viral, microbial, proteins and other Antigens in normal histological dyed fabrics are not visible. Antibodies in the sera bear the label of either fluorochrome, i.e. dye, glowing in the dark field (in other words, giving fluorescence)or a coloring enzyme. If the antigen is in the investigated tissues, resulting complex antigen-antibody plus marker pinpoints its presence, localization, intensity of staining, will help to determine the type of tumor, to study its properties. The most common immunoassay method. Antibodies ink serum are not fluorochrome, and the enzyme horseradish peroxidase, rarely another enzyme, such as alkaline phosphatase. There are several options, the ants specified method. The most commonly used two of them - peroxidase-anti-peroxidase (PAP-method PAP method) and the method avidin-bioteknologi complex (ABC-method, ABC method) (Immunocytochemistry. A Practical Approach. Ed. by J.E.Beesley. Department of Pharmacology, Wellcome Research Laboratories, Beckenham, Kent / Oxford University Press, 1993, 248; Dodson, A., Campbell F., Biotin inclusions: a Potential Pitfall in immunohistochemistry (letter). Histopathology, 1999; 34; 178-179; UK NEOAS Immunocytochemistry News. J. Cell. Pathol. 2001; 5:189; Guide immunohistochemical diagnosis of human tumors. Edited Svitava, Nutricline. Kazan "Title", 2004, 451; Diagnostic Immunohistochemistry/Td. by D.Dabbs. Churchill Livingstone, 2006, 828). When PAP enzymatic method, i.e. peroxidase, the antibody binds to the primary antibody in the antigen by another antibody-bridge. As would be deployed to connect the links to their short immunoglobulin chains to bind them in a long chain. Thus, there are 3 layers of substances: antigen with immobilized antibody in the tissue, the antibody-bridge and PAP complex, in which the molecules of peroxidase are located between two short chains of two coupled antibodies carrying the enzyme.

The use of high quality reagents, simplification and automation of immunohistochemical studies have made this method a necessary tool for solving diagnostic issues facing pathologists.

There is a method of staining of cartilage and bone is Kani (patent RF №2033610, 20.04.1995) by impact dyes on histological slice, rinse in distilled water, dehydrated, enlightenment and conclusions in balm, characterized in that the additional stages provide counterstaining of 0.1%alcian blue, cooked on the buffer with a pH of 4.4 for 20-45 min at 37°C in thermostat and eosin, while eosin counterstaining is performed at room temperature. The disadvantages of this method include its duration, making it difficult to use it for intraoperative diagnosis.

The known method immunohistochemical detection of protein on paraffin histological sections (patent RF №2098825, 10.12.1997), including the dewaxing, dehydration, blocking of endogenous peroxidase and then nonspecific adsorption of immunoglobulins, processing of histological sections of primary antibodies followed by incubation of secondary antibodies, the expression of the peroxidase activity, colouring and detection of proteins by the presence of brown staining, characterized in that prior to the processing of histological sections of primary antibodies in their solution further added polyethylene glycol 6000 to the final concentration in the solution is 5%.

It is known that immunohistochemical staining of paraffin tissue sections takes about 4-5 hours (depending the spine of the characteristics of the reagents). Color cryostatic slices eliminates the time-consuming steps of dewaxing, dehydration, damascenone antigens, rehydration, which greatly speeds up the procedure. However, the remaining processing steps fabrics take on average 75-80 minutes, which is not suitable for intraoperative diagnosis.

Peter Ruck (Peter Ruck. EnVision™ for rapid immunostaining in intraoperative frozen section diagnosis. Institute of Pathology, University of Tubingen, Germany, 2001 Institute of Pathology, University of Tubingen, Germany, 2001) proposed a Protocol for immunohistochemical staining cryostatic sections: how to paint Quick intraoperative immunohistochemical diagnosis on frozen sections, allowing to receive the result in 12-13 minutes.

Table 1
"Step-by-step description of the methodology P.Ruck: prototype method
Stages immunohistochemical stainingThe incubation periodTemperature, °C
1. Fixing cryostatic tissue slices in acetone and drying slices1 min 15 secRoom 25°C
2. Incubation with primary antibodies3 min37°C
3. Washing buffer (Tris-buffer, pH 7.4)1025°C
4. Incubation with secondary antibody (EnVision™complex Dual Link)3 min37°C
5. Washing buffer (Tris-buffer, pH 7.4)1025°C
6. Incubation with substrate-Chromogen3 min37°C
7. Rinsing with tap water1025°C
8. Rinsing with distilled water525°C
9. Contrasting with hematoxylin Mayer1525°C
10. Rinsing with tap water3042°C
11. Conclusion balm3025°C

Immunohistochemical staining of serial cryostatic of tissue sections was performed peroxidase method using mono - and polyclonal antibodies and systems the visualization EnVision™ (DAKO). Working concentrations of the primary antibodies in 4-10 times the concentration recommended by the supplied instructions. For cultivation of primary antibodies used solvent antibodies, reducing background staining (DAKO). All stages of staining was held on the heating table at 37°C. the Total reaction time (without microscopy and assessment of the obtained results): 12 min 05 C. due to the time needed for preparation cryostatic sections for microscopy and assessment of the obtained results the total time varies between 20-22 minutes.

This method adopted by us for the prototype. The main disadvantage of the prototype is that the duration of the study exceeds 12 minutes while one of the main tasks of morphological research method is a lifetime diagnosis of pathological processes in biopsy and surgical material and, above all, diagnosis cristatum cut during surgery. Intraoperative morphological diagnosis is a very difficult task even for experienced professionals, as it occurs in conditions of lack of time (12-15 minutes) and is based solely on the shape of the tissue structures, cell sizes, etc. that does not exclude the heuristic error. However, from the initial morphology is ical diagnosis depends tactics of surgical treatment, identifying opportunities conserving surgery, the determination of the level of tissue excision, etc. In this regard visokooktanovim is the introduction of intraoperational morphological diagnosis methods immunohistochemical examination of tissues, allowing more quickly and accurately determine the histogenesis of the tumor, its malignant potential, identifying metastases to evaluate the status of the margins of resection, the resection margins of the tumor cells, etc.

The objective of the invention is to increase the efficiency of intraoperative diagnosis due to the acceleration of immunohistochemical staining of tissue cryostatic sections for further microscopic examination.

This object is achieved by the fact that carry out all the necessary stages of dyeing: fixing slices in acetone, the impact of primary and secondary antibodies to the antigenic structure of the tissue, rinsed in Tris-buffer pH 7.4 and incubation with substrate-Chromogen solution, rinse with distilled water, contrasting with hematoxylin and conclusion in balm. But incubation of tissue slices with a mixture of primary and secondary antibodies is carried out simultaneously, which first cuts applied primary antibodies, and 15 seconds of incubation (not washing buffer) applied secondary antibodies; for incubation with substrate-Chromogen used with useprivatekey solution of substrate-Chromogen directly in front of his application; washing is carried out with the buffer solution and distilled water, heated to a temperature of 37°C. All of the above techniques can significantly reduce the time immunohistochemical reactions. Thus, our proposed departure from the standard method due to the need to minimize the time of the study the tumor tissue at the time of surgery when surgeons are forced to cancel the operation and wait at the operating table results immunomorphological studies that determine further tactics of surgical treatment.

Immunohistochemical staining provides a unique opportunity to assess the biological potential of tumor cells: growth rate, the prognosis of the tumor process, response to chemotherapy and hormonal treatment. There is a high degree of reliability when the tissues in determining the phenotype of a tumor, and in some cases tissues eliminates the need for other more costly and time-consuming diagnostic procedures.

The inventive method allows to use a fairly wide range of antibodies. We used markers: Cytokeratin (AE/E), Cytokeratin 7 (OV-TL 12/30), Vimentin (V9), PLAP (8A9), LCA (2B11+PD7/26), S-100, Melanosom (HMB45), Synaptophysin(SY38), CD30 (Ber-H2), Ki-67 (MIB-1), P63 (4A4), Cytokeratin 34bE12 (34bE12), CA-125 (Ov185:1), PSA (ER-PR8), Galectin-3 (9C4), p504S (13H4), p53 (DO-7) (DAKO).

The first series of our ASCS is adavani was dedicated intraoperative study of the corresponding tissues we have developed a method of rapid immunohistochemical staining cryostatic of tissue sections.

In the second series of studies was conducted postoperative immunohistochemical study of the corresponding tissues in paraffin sections using the standard method with the same markers and reagents visualization. Comparative analysis of the results of the staining showed a complete coincidence immunophenotypic characteristics of tumor cells, detected as cryostatic and on paraffin sections. Variable was only the intensity of the color.

In the study we have developed a method of immunophenotyping in cryostatic tissues lasts 7-8 minutes. Details of the Protocol are presented in table 2.

Table 2
"Step-by-step description of the claimed methods immunohistochemical staining cryostatic of tissue sections in terms of intraoperative diagnostics
Stages immunohistochemical stainingThe incubation periodTemperature, °C
1. Fixing cryostatic slices in acetone50Room
25°C
2. Drying slices 1037°C
3. Incubation with a mixture of primary and secondary antibodies (EnVision™complex)4 min37°C
4. Washing buffer (Tris-buffer pH 7.4)1037°C
5. Incubation with substrate-Chromogen1 min37°C
6. Rinsing with distilled water1037°C
7. Contrasting with hematoxylin Mayer1525°C
8. Rinsing with tap water1542°C
9. Clucene in balm1525°C

Total reaction time: 7 min 05 C.

New techniques, in contrast to methods P.Ruck are:

1. The temperature of drying to 37°C, which reduces the time of drying after fixation in acetone.

2. The elimination of the washing step with buffer solution after incubation with the primary antibody by simultaneous incubation of tissue slices with a mixture of Erwinia and secondary antibodies (i.e. items # 2 and # 4 methodology P.Ruck combined into one item, and item No. 3 methods P.Ruck cancelled). Thus shortens the time of incubation with primary and secondary antibodies.

3. Originally applied primary antibodies on tissue sections, and after 15 seconds of incubation (not washing buffer) applied secondary antibodies. The total incubation time at this stage - 4 minutes

4. Use a freshly prepared solution of substrate-Chromogen immediately before its application, thereby decreasing the incubation time with substrate-Chromogen from 3 min to 1 min

5. The use of buffer solution and distilled water, heated to a temperature of 37°C, thereby reducing the time of washing the tissue slices after incubation.

6. After incubation with hematoxylin time is reduced flush with water for 15 s and detention in balm 15 C.

Comparative analysis with the prototype showed that the proposed method has significant differences from the known; as a result, the execution time of the immunohistochemical reaction on frozen sections is reduced by 5 min, in terms of intraoperative diagnosis is essential. This allows to conclude that the criteria of "novelty", "significant differences" and "industrial applicability".

Detailed description of the method and examples of its practical implementation.

And ucaut serial cryostate the tissue sections, stained with hematoxylin and eosin, and produce immunohistochemical staining of peroxidase method using mono - and polyclonal antibodies and visualization system is EnVision™ (DAKO, Denmark). The choice of the visualization system is defined by its high sensitivity and the two-step procedure, painting, taking less time than the ABC-method. Working concentrations of the primary antibodies in line with the recommendations enclosed instructions or exceed 2-10 times. For cultivation of primary antibodies used solvent antibodies, reducing background staining (DAKO). All stages of the dyeing carried out on the heating table at 37°C.

On cryostatis fabric cut, fixed in acetone for 50 seconds original primary cause, and after 15 seconds of incubation (not washing buffer) secondary antibody and incubated for 4 minutes at 37°C, followed by staining of the substrate-Chromogen and contrast with hematoxylin. A solution of substrate-Chromogen prepared immediately before its application. For washing the tissue sections using a buffer solution and distilled water, heated to a temperature of 37°C.

Examples of work with the tissue of the prostate:

Example 1. Patient D. Immunohistochemical staining Cristallago cut tissue carcinoma of the prostate with antic the Lamy to high molecular weight cytokeratins, clone 34β12, magnification ×10 (figure 1). Figure visible positive membrane staining (brown) of the basal cells of non-neoplastic glands with antibodies to high molecular weight cytokeratins, clone 34β12 and negative painting with the same antibodies at the site of tumor glands that is immunomorphological diagnostic sign of adenocarcinoma of the prostate. The research time is 7 minutes.

Example 2. Patient S. Immunohistochemical staining Cristallago cut tissue of the prostate with antibodies to high molecular weight cytokeratins, clone 34βE12, magnification ×20 (figure 2). Figure visible positive membrane staining (brown) of the basal cells of non-neoplastic glands with antibodies to high molecular weight cytokeratins, clone 34β12. Areas of adenocarcinoma in the investigated material no. The research time - 7 minutes 4 C.

Example 3. Patient A. Immunohistochemical staining Cristallago cut tissue carcinoma of the prostate with p504S antibody (clone N), magnification ×40 (figure 3). Figure visible positive cytoplasmic staining (brown) part of tumor cells with antibodies to p504S (clone N), which is a sign of malignancy. The research time - 7 minutes 6 C.

Example 4. Patient I. Immunohistochemical staining Cristallago cut tissue tumors of the ovary with antibodies to CA-125 (cloe Ov185:1), magnification ×20 (figure 4). Figure visible positive cytoplasmic staining (brown) of tumor cells with antibodies to CA-125 (clone Ov185:1), indicating that the origin of the tumor from ovarian tissue. The research time - 7 minutes 5 C.

Example 5. Patient A. Immunohistochemical staining Cristallago cut tissue tumors of the lung with antibodies to cytokeratin 7 (clone OV-TL 12/30), magnification ×20 (figure 5). Figure visible positive membrane staining (brown) of tumor cells with antibodies to cytokeratin 7 (clone OV-TL 12/30). The research time is 7 minutes.

Example 6. Patient R. Immunohistochemical staining Cristallago cut tissue tumors of the colon with antibodies to cytokeratin (clone AE/AE), magnification ×20 (6). Figure visible positive cytoplasmic staining (brown) of tumor cells with antibodies to pantisocracy (clone AE/AE). The research time - 7 minutes 6 C.

Our studies were performed on the operating material Regional clinical hospital №1 named after. Professor Svechenovskoj Krasnodar. Researched remote parts of the lungs, bronholitin, biopsy of tumors of the mediastinum, tissue resection of the prostate tumor tissue of the prostate tissue of intestinal tumors, ovarian cancer, thyroid cancer, obtained from 73 patients were treated from October 2007 to March 009, The results and the quality of immunohistochemical staining cryostatic and paraffin tissue sections were identical in all cases.

Thus, as a result of research we have developed a rapid method of immunohistochemical staining for cryostatic sections, allowing to reduce the staining procedure on average 7-8 minutes, which makes it relevant to its wide use in intraoperative diagnosis.

The way immunohistochemical staining cryostatic of tissue sections in terms of intraoperative diagnosis by fixing the slices in acetone, the impact of primary and secondary antibodies on tissue slice, rinse in Tris buffer pH 7.4 and incubation with substrate-Chromogen solution, rinsing with distilled water, contrasting with hematoxylin and conclusions in balm, characterized in that conduct simultaneous incubation of tissue slices with a mixture of primary and secondary antibodies, which are initially in tissue sections is applied primary antibodies, and after 15 incubation, without rinsing buffer, causing secondary antibodies and conduct co-incubation for 4 min, a solution of the substrate-Chromogen incubation prepared immediately before application, the washing of the slices is performed by the buffer solution and distilled water, heated to a temperature of 37°C.



 

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1 tbl

FIELD: physics.

SUBSTANCE: proposed method comprises dipping magnetostrictive element into controlled fluid. Then, constant magnetising field and variable magnetic fields are induced in magnetostrictive element area to excite lengthwise oscillations of the latter and variable magnetic field is cut off. Now, resonance frequency of signal generated in coil by magnetostrictive element is determined. Then, fluid viscosity is calculated. Besides, prior to calculating fluid viscosity, magnetostrictive element temperature is controlled and compensation for temperature dependence of magnetostrictive element intrinsic oscillation frequency is performed.

EFFECT: higher accuracy of prompt control and reliability of operation, reduced overall dimensions.

10 cl, 6 dwg

FIELD: machine building.

SUBSTANCE: procedure for measuring viscosity of liquids is designed for control of liquids of high viscosity in chemical, food and other branches of industries. The procedure for measuring viscosity of liquid consists in effecting surface of liquid with a gas jet of alternate velocity and in generation of forced surface-mode waves. Further, there are changed and measured frequency of fluctuations of gas jet velocity. Velocity of gas jet is changed according to a harmonic law. There is measured phase-shifting between fluctuations of gas velocity and liquid surface waves. Viscosity is determined on base of frequency of gas jet velocity fluctuations when phase-shifting reaches a specified value.

EFFECT: upgraded accuracy of viscosity measurement; simplified design of procedure.

3 dwg

FIELD: machine building.

SUBSTANCE: there are excited bending self-oscillations of reference and analysed cantilevers. Also, for determination of coordinate, length and depth of open fracture of flexible cantilever there are measured the first three frequencies Ω1, Ω2, Ω3, and Ω1*, Ω2*, Ω3* of self-oscillations of the reference and analysed cantilevers correspondingly. Further, from the frequency equation made for a model beam without a fracture, there is determined value of its first three frequencies ω1, ω2, ω3 of self-oscillations. Through ratio of values of corresponding frequencies of oscillations of the model and reference cantilevers without fractures there are found three frequency coefficients k1, k2, k3 of reference of model correction: k111, k222, k333, there are multiplied measured values of the first three frequencies Ω1*, Ω2*, Ω3* of self-oscillations of analysed cantilever with an open fracture to corresponding frequency coefficients of correctness, and there are found exactly the values of the first three frequencies ω1*=k11*, ω2*=k22*, ω3*=k33* of self-oscillations used in the frequency equation on the model cantilever with a cut for calculation of its coordinate, length and depth corresponding to coordinate, length and depth of open fracture of analysed flexible cantilever.

EFFECT: determination of coordinate and dimension of open fracture of flexible cantilever.

4 dwg

FIELD: automatical aids for sampling liquids.

SUBSTANCE: system for sampling and delivering filtrate has filter submerged into tested medium and connected with collecting tank and vacuum pressure source which is connected with top hole of collecting tank by means of pneumatic pipe. System has sample receiving tank connected with collecting tank and control unit which has first output to be connected with vacuum pressure source. Collecting tank has two separated chambers - washing chamber and dispatching chamber. Lower hole of washing chamber has to be lower hole of collecting tank and side hole of dispatching chamber has to be side hole of collecting tank. Floating valve is installed inside washing chamber to shut off lower and top holes. Filter is connected with lower hole of collecting tank through sampling pipe. Side hole of collecting tank is connected with lower hole of tank for receiving samples through sampling pipe. Flow-type sensor and check valve are installed inside transportation pipe. Output of flow-type sensor is connected with input of control unit; second output of control unit is connected with control input of analyzer.

EFFECT: improved precision of measurement of sample ion composition; prolonged service life of filter.

1 cl, 1 dwg

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