CN101275889A - Target tissue carbon core multidimensional positioning method and product in tissue wax stone - Google Patents

Abstract

Disclosed is a multidimensional positioning method of target tissue carbon core and the product in the paraffin-embedded tissue block, whereby the target tissue in the paraffin-embedded tissue block can be accurately found, which improves the efficiency of preparing the tissue chip.

Description

The method and the product of destination organization carbon core multidimensional location in the paraffin embedded tissues

One, technical field

The present invention relates to a kind of contrast haematoxylin Yihong stained, accurate localizing objects histocyte and drill through the method for organizing core in paraffin embedded tissues more specifically, relates to the method that destination organization carbon core location technology in a kind of paraffin embedded tissues accurately obtains tumor tissues.

Two, background technology

Along with histology, pathological development, people are to human body or other vegeto-animal understanding, and the organ level on anatomy develops to tissue, cellular level.But because complete organ is difficult to use microscopic examination, so need be cut into the very thin section observation of dyeing to organ,, histological techniques occur in order to address this problem.The principle of this method is by tissue that organ is cut into small pieces, after formalin fixed, utilize alcohol to slough moisture in this tissue, immerse dimethylbenzene again or benzole soln is sloughed alcohol, last piece of tissue immerses in the melted paraffin solution sloughs dimethylbenzene, put into mould and carry out embedding with paraffin solution, the cooling back forms paraffin mass.This paraffin mass utilizes microtome to cut into slices, and wax disk(-sc) is mounted baked sheet, dewaxing again on glass sheet, just can carry out observational study with microscope after dyeing, the mounting.At present, histological techniques is not only simple dyeing microscopic examination, various specific stains, immunohistochemistry technology, hybridization in situ technique, original position round pcr have also occurred.

In recent years along with genomics, protein science research and development, the physiology of people's research and disease progressively from molecular level to cellular level and organize the level development, therefore, how to utilize and organize research, be will one of deal with problems in the research at present.Adopt tradition to organize the Study on Technology method, because the tissue specimen requirement is big, the experimental work amount is big, and be difficult to adapt to research is wanted.In recent years, the tissue micro-array chip technology occurs, solved the problems referred to above.This technology fitly is arranged in dozens of, a hundreds of and even thousands of little tissue on a certain carrier (normally microslide) and is made into the micro histotomy, and its fundamental procedure (as shown in Figure 1) comprising: the relevant position, the organization chip that design and draft the making of the taxonomic series of organization chip, the pathological diagnosis of checking tissue or diseases related, tissue positioned, array wax stone, drill through tissue and transfer to blank paraffin mass again from the source wax stone are cut into slices.The characteristics of organization chip have: volume is little, information content is big, high flux; Reduce or avoided destruction the original structure wax stone; The accurate positioning of tissue samples reduces invalid tissue; Batch making and possible interpretation of result robotization; The processing of the case of separate sources, dyeing and analysis condition are identical.

The invention of tissue array technology has brought very big promotion for histology, pathological development.The difficult point of this technology is exactly tissue positioned and drills through from source pathology wax stone and to organize core, and " invalid tissue " problem will appear in this step misoperation." invalid tissue " problem is meant that final organization chip is carried out the further consultation of bush Su Yi red colouring pathology confirms that every bit is whether consistent with the required types of organization of array design, if this is the tumor tissues of interstitial fibers vascular tissue rather than planned, then this some tissue just is that " invalid tissue " can not be analyzed by test, if " invalid tissue " accounts for and counts morely in the organization chip, will greatly reduce the using value of organization chip.Cause its main cause of " invalid tissue " to be, paraffin embedded tissues is when making stained, paraffin mass is earlier-20 ℃ of refrigerator precoolings, carry out the paraffin organization section then, its thickness has only 4～6 microns extruding that are subjected to blade can produce in various degree contraction, place the amplification that the section of 39 ℃～44 ℃ higher water temperatures exhibition sheets again can be in various degree again, the tissue morphology size is variant in last like this stained of making and the paraffin, the pathology expert diagnoses on stained and the scope of marked tumor tissue, compare the position of determining tumor tissues with reference to the tissue in scope of drawing a circle to approve on the stained and the pathology wax stone, at this moment just be easy to take place error the excluded nonneoplastic tissue of script is selected into.

Three, summary of the invention

The purpose of this invention is to provide a kind of efficiently, easy, low-cost, utilize carbon core tissue positioned technology accurately to obtain the method for destination organization, to overcome the weak point in above-mentioned.Main design of the present invention is, be organized in and only account for a part in the wax stone and all be blank paraffin on every side, on the position that blank paraffin is fixed, place the carbon core of 3～5 different-diameters, these carbon cores do not influence histotomy, if tissue is squeezed and is out of shape so that the carbon core of fixed position also can produce identical distortion when cutting into slices, after being made into the HE stained, the pathology further consultation is irised out the target tumor tissue with marker pen thereon, according to irising out the position to the distance of location carbon core and the location variable of carbon core, by artificial estimation or computer picture scanning collection software HE staining section and paraffin mass are compared and to be easy on paraffin mass, determine the position of target tumor tissue, so just can get the target tumor tissue accurately, greatly improve the product percent of pass of organization chip.

The preparation method of charcoal core: at first select the high-quality charcoal, be cut to the bulk of 15 * 15 * 15 millimeters sizes, putting into the tissue dewatering frame then dewaters according to conventional organization, transparent, the waxdip embedding program is handled, promptly by following reagent flow processing: 1. anhydrous alcohol 1 cylinder is 1 hour, 2. anhydrous alcohol 2 cylinders are 2 hours, 3. dimethylbenzene 1 cylinder is 1 hour, 4. dimethylbenzene 2 cylinders are 2 hours, 5. in 62 degree paraffin 1 cylinders 2 hours, 6. in 62 degree paraffin 2 cylinders 2 hours, be made into the charcoal wax stone at last on the paraffin embedding machine, use the charcoal core of the hollow needle making different-diameter of different-diameter, the diameter of charcoal core commonly used is 0.6 millimeter, 1.0 millimeter, 1.5 millimeter, 2.0 millimeters, 2.5 millimeters or 3.0 millimeters.The charcoal core that these perform can be used for the location of piece of tissue target tumor tissue, and the method for placing the carbon core on paraffin embedded tissues can have following 2 kinds:

1, mould plantation embedding carbon core method

Its inside surface of stainless steel mould that the normal tissue embedding is used is smooth, can handle this stainless steel mould, can make 3-5 groove respectively on four of the portion bottom surface angles within it, is generally four grooves.Aforementioned four grooves, the distance of each groove distance both sides frame is respectively 3 millimeters, and the degree of depth of this groove can be a numerical value between 0.5 millimeter to 2 millimeters, the diameter of groove can for: 0.6 millimeter, 1.0 millimeters, 1.5 millimeters, 2.0 millimeter, 2.5 millimeters or 3.0 millimeters.As shown in Figure 2: (2) 2 of 1.0 millimeters grooves of diameter are arranged on this stainless steel mould (1), 1.5 (3) 1 of millimeter grooves, 2.0 (4) 1 of millimeter grooves, totally 4 grooves, and 3 millimeters on each groove distance frame, the degree of depth is 1.0 millimeters, when embedding, earlier the carbon core of length (being generally 5 millimeters) appropriate diameter is put into corresponding groove, put into the good tumor tissue of processed then, put the dehydration frame of plastic, perfusion paraffin, cooling is taken out wax stone from stainless steel mould, being made into one has as shown in Figure 3 had the paraffin embedded tissues of fixed position carbon core.

2, the core substitution method is got in the paraffin embedded tissues location

The tumor tissues wax stone can use commonsense method to make, can carry out secondary processing with the tissue array instrument after making, blank wax place around tissue implants the carbon core accurately, 2 in 1.0 millimeters carbon cores of diameter in a pathological tissue wax stone, have been inserted respectively as Fig. 3 place, 1.5 1 in millimeter carbon core, 1 in 2.0 millimeters carbon cores, totally 4 carbon cores, also can use the fixing frame of plastic of length to determine carbon core position, the carbon core is implanted in punching.Put into the baking ovens baking 4 hours that paraffin embedded tissues behind the carbon core also will place 51 degree, carbon core and tissue array are closely merged.

Just can carry out paraffin section after adopting above-mentioned two kinds of methods to make the tumor tissues wax stone of putting into the charcoal core according to the common pathology operation, the dyeing of haematoxylin Yihong, the mark of pathological diagnosis and target tumor tissue, on stained, calculate the distance that target tumor is organized into four charcoal cores, just can easily accurately calculate the position of target tumor tissue in the paraffin embedded tissues, thereby guarantee the accuracy that core is got in the organization chip making.

Description of drawings:

Fig. 1 organization chip is made basic procedure

The stainless steel mould of Fig. 2 band groove

The paraffin embedded tissues of the good charcoal core of Fig. 3 mark

Should be understood that the exercise question of this specification and the embodiment that this specification is addressed only are used for explanation The present invention and being not used in limits scope of the present invention.

Claims (10)

1. the method for destination organization carbon core multidimensional location in the paraffin embedded tissues is characterized in that may further comprise the steps:

A) preparation charcoal piece wax stone;

B) in charcoal piece wax stone, drill through the charcoal core;

C) the charcoal core is embedded into and organizes paraffin mass, preparation charcoal core position tissue wax stone;

D) charcoal core position tissue wax stone section statining is the coordinate setting destination organization with the charcoal core.

2. method according to claim 1 is characterized in that described preparation carbon piece paraffin mass may further comprise the steps:

A) choose 15 * 15 * 15 millimeters charcoal piece;

B) carbon piece preparation carbon piece wax stone after dehydration, transparent, waxdip, embedding.

3. method according to claim 1 is characterized in that the charcoal core diameter of being addressed can be 0.6 millimeter, 1.0 millimeters, 1.5 millimeters, 2.0 millimeters, 2.5 millimeters or 3.0 millimeters, and length is 3 to 15 millimeters.

4. method according to claim 1 is characterized in that described charcoal core is embedded into to organize paraffin mass may further comprise the steps:

A) the charcoal core is implanted in the groove of the embedding mould that is provided with groove;

B) at the other tissue for the treatment of embedding of putting into of charcoal core, perfusion paraffin.

5. method according to claim 1 is characterized in that described charcoal core is embedded into to organize paraffin mass may further comprise the steps:

A) organizing the blank wax place punching on every side of paraffin mass tissue;

B) above-mentioned aerial implantation charcoal core;

C) paraffin embedded tissues behind the implantation carbon core toasts.

6. method according to claim 5, the condition that it is characterized in that described baking are 51 degree, 4 hours.

7. method according to claim 4 is characterized in that the embedding mould bottom surface of being addressed that is provided with groove is provided with 4 grooves that the degree of depth is any one numerical value between 0.5 millimeter to 2 millimeters.

8. embedding mould according to claim 7 is characterized in that the groove diameter of being addressed can be 0.6 millimeter, 1.0 millimeters, 1.5 millimeters, 2.0 millimeters, 2.5 millimeters or 3.0 millimeters.

9. embedding mould according to claim 8 is characterized in that 4 grooves being addressed are positioned at four angles of embedding frame bottom surface, 3 millimeters on each groove distance frame.

10. charcoal core multidimensional position tissue wax stone, comprise the tissue in paraffin and the embedding, it is characterized in that in the described wax stone, be embedded with 3 to 5 charcoal cores, the charcoal core diameter can be 0.6 millimeter, 1.0 millimeters, 1.5 millimeters, 2.0 millimeters, 2.5 millimeters or 3.0 millimeters, and length is 5 millimeters.

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