CN100460850C

CN100460850C - Making method of body fluid cell lump paraffin section

Info

Publication number: CN100460850C
Application number: CNB2005100212966A
Authority: CN
Inventors: 徐华
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-07-20
Filing date: 2005-07-20
Publication date: 2009-02-11
Anticipated expiration: 2025-07-20
Also published as: CN1737526A

Abstract

This invention relates to body liquid cell olefin slice making process, which comprises pre-processing, decenter deposition and fixing dehydrate wax impregnation, burying, slicing and HE dying and sealing. The invention works together with daily work with clear background of HE and immune organization dying without dark background of agar burying and processing the molecule burying and improves the accuracy of the body liquid cell diagnose.

Description

Making method of body fluid cell lump paraffin section

Technical field

The present invention is that clinical medicine detects one of tumour cell and cellular elements mark detection method.

Background technology

The cell of human body is under the effect of carcinogenic factor, the change of matter can take place and become cancer cell in the character of cell gradually, its cell is under the influence of long-term carcinogenic factor, the formed tumour cell of cell just develops into malignant tumour when being increased to some, in order to make a definite diagnosis the state of an illness, so that suit the remedy to the case, strive for best treatment, time and effect, one of method that present medical department adopts the patient, be to search cancer cell in the body fluid, the general hydro-extractor that uses is made the centrifugal heavy smear of deciding, or use agar embedding cell lump paraffin section, use smear hydro-extractor simple and fast, but equipment is more expensive, can not be used for the immunohistochemical staining inspection, can not carry out the cellular elements markers tests, can not detect more accurately tumour patient, meet difficult smear on occasion, be difficult to tumor type is classified, sometimes be difficult to distinguish the tumour cell of regression or the mesothelial cell of hyperplasia regression, use agar cell lump specimens paraffin embedding slices program more, need to make in advance agar again and comparatively bother, so need to improve.

Summary of the invention

It is simple to the purpose of this invention is to provide a kind of method, is difficult for making mistakes the making method of body fluid cell lump paraffin section that the result is stable.

Making step of the present invention is as follows:

1) centrifugation pre-treatment: with its ratio of formaldehyde of body fluid adding 40% is body fluid: 40% formaldehyde equals 9:1, after static 5 minutes to 5 hours, adds NaCl and makes the interior NaCl concentration of body fluid reach 0.9%, precipitates 30 minutes to 72 hours;

2) centrifugation: be injected into 5ml-20ml centrifuge tube after above-mentioned sediment shaken up and carry out centrifugal treating, centrifuge speed is 3000-5000 rev/mins, each time is 5-15 minutes, discard upper clear supernate after each centrifugal treating, sediment reinjects, carry out 2-8 times repeatedly, up to obtaining more cell quantity;

3) fixing: as the product of above-mentioned centrifugation to be discarded upper clear supernate inject 10% neutral formalin, fix 2 hours.Histiocytic structure is kept, keep the plesiomorphism when cell is with life in the cell lump, prevent aqtocytolysis;

4) dehydration, transparent, waxdip: the sediment after the said fixing processing is wrapped up with double gauze, automatic dehydration dewaters in biological tissue, or adopt manually and dewater, the alcohol temperature is 45 degree Celsius during dehydration, 75% dehydration of alcohol 60min, 85% dehydration of alcohol 120min, 95% dehydration of alcohol 120min, the absolute ethyl alcohol I 90min that dewaters, the absolute ethyl alcohol II 90min that dewaters, the absolute ethyl alcohol III 90min that dewaters, adopting has water wettability to have lipophilic dimethylbenzene or TO transparent agent to carry out transparent, waxdip again, reaches the purpose that paraffin immerses cell;

5) embedding: with the cooling of the cell lump behind the waxdip, pulverizing, pulverize the back mixing, put into the paraffin embedding frame bottom, condense together, slowly dissolve paraffin and make the cell composition more even like this in cell lump inside along the adding of embedding edge edge, embedded easily;

6) section dewaxing: slice thickness is 3-5 microns.The concrete steps that dewax are as follows: the 50min that in thermostatic drying chamber, dewaxes that 1. cuts into slices, and temperature is 60 degree Celsius; 2. dimethylbenzene I dewaxing 10min; 3. dimethylbenzene II dewaxing 5min; 4. absolute ethyl alcohol flush away dimethylbenzene 1min * 2; 5. 6. 7. 85% alcohol 1min of 90% alcohol 1min of 95% alcohol 1min;

7) HE dyeing: washed 1-3 minutes in flowing water or the hydrostatic in the section that will dewax earlier, dye routinely, to the chemical staining of parts of fine mass section can carrying out as required immuning tissue, its method is identical with common immunohistochemical staining, just can carry out the cellular elements markers tests;

To adopt neutral resins glue mounting to finish manufacturing process at last.

The present invention makes section with other and compares, method is simple, operation steps is few, be difficult for making mistakes, the result is stable, do not need special material and facility, can carry out with usual routine work, the section background is clear, and colour developing is good, effect is satisfied, the sort of blurred background that does not have the agar embedding can be carried out the work of molecular pathology aspect, carries out the cellular elements markers tests, improved the accuracy rate of body fluid cell pathological diagnosis, improved range of application,, the result of treatment of clinical patient provided treat guiding suggestion more accurately particularly to the detection of drug resistant gene representation.

Embodiment

The present invention detects 50 routine ascites pleural fluid and compares with common centrifugal smear results, look into and see cancer cell 5 examples, there is not significant difference at tumour cell aspect detecting, but the tumour cell that the present invention obtains is more, can carry out immunohistochemical staining, the background of immunohistochemical staining is clear, colour developing is good, particularly can carry out the detection of drug resistant gene expression product, chemotherapy of tumors had important help, and can carry out the tumour cell classification by immunohistochemical staining and improve quality of diagnosis, carry out cell marker and detect, be difficult to the district sometimes respectively, use the type of immunohistochemical staining labeled cell for the mesothelial cell of regression and the tumour cell of regression, can improve the accuracy of diagnosis, can carry out staging and provide treating guiding suggestion more accurately.

Claims

1. the method for a making method of body fluid cell lump paraffin section, its step is as follows:

1) centrifugation pre-treatment: body fluid is added 40% formaldehyde after static 5 minutes to 5 hours, add NaCl and make that NaCl concentration reaches 0.9% in the body fluid, precipitate 30 minutes to 72 hours:

2) centrifugation: be injected into the 5ml-20ml centrifuge tube after above-mentioned sediment shaken up and carry out centrifugal treating, centrifuge speed is 3000-5000 rev/min, each time is 5-15 minute, abandoning supernatant after each centrifugal treating, sediment reinjects, carry out 2-8 time repeatedly, up to obtaining more cell quantity;

3) fixing: as the product of above-mentioned centrifugal sediment to be discarded upper clear supernate inject 10% neutral formalin, fix 2 hours;

4) dehydration, transparent, waxdip: the sediment after the said fixing processing is wrapped up with double gauze, adopt biological tissue's automatic dehydration to dewater, or adopt manually and dewater, the alcohol temperature is 45 degree Celsius during dehydration, 75% dehydration of alcohol 60min, 85% dehydration of alcohol 120min, 95% dehydration of alcohol 120min, the absolute ethyl alcohol I 90min that dewaters, the absolute ethyl alcohol II 90min that dewaters, the absolute ethyl alcohol III 90min that dewaters, adopting has water wettability to have lipophilic dimethylbenzene or TO transparent agent to carry out transparent, waxdip again, reaches the purpose that paraffin immerses cell;

5) embedding: with the cooling of the cell lump behind the waxdip, pulverizing, pulverize the back mixing, put into the paraffin embedding frame bottom, condense together, slowly dissolve paraffin and make the cell composition more uniform embedded like this in cell lump inside along the adding of embedding edge edge;

6) section dewaxing: slice thickness is the 3-5 micron, and the concrete steps that dewax are as follows: the 50min that in thermostatic drying chamber, dewaxes that 1. cuts into slices, and temperature is 60 degree Celsius; 2. dimethylbenzene I dewaxing 10min; 3. dimethylbenzene II dewaxing 5min; 4. absolute ethyl alcohol flush away dimethylbenzene 1min carries out twice repeatedly; 5. 95% alcohol 1min; 6. 90% alcohol 1min; 7. 85% alcohol 1min;

7) HF dyeing: the section that will dewax was earlier washed 1-3 minute in flowing water or hydrostatic, dyeed routinely, and the parts of fine mass can carry out the chemical staining of immuning tissue as required;

To adopt the neutral gum mounting at last, finish manufacturing process.