CN104359746B - Cell mass collector and the method for collecting cell in humoral specimen - Google Patents
Cell mass collector and the method for collecting cell in humoral specimen Download PDFInfo
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- CN104359746B CN104359746B CN201410661633.7A CN201410661633A CN104359746B CN 104359746 B CN104359746 B CN 104359746B CN 201410661633 A CN201410661633 A CN 201410661633A CN 104359746 B CN104359746 B CN 104359746B
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- centrifuge tube
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Abstract
A kind of cell mass collector and the method for collecting cell in humoral specimen, collector includes the first centrifuge tube, the support tube of tubular is provided with first centrifuge tube, the second centrifuge tube stretched into support tube provided with lower end in the first centrifuge tube conical section, the conical section of second centrifuge tube is provided with multiple fluid holes, in use, being put into filter paper in the conical section of the second centrifuge tube, filter paper is set to be bonded with the inwall of the conical section.Formalin is added in humoral specimen, sediment is obtained, centrifugation, adds ethanol and protein chelate agent, mix, it is filled on filter paper, centrifuges, cell mass is formed on filter paper, embed, be dehydrated by existing method, being embedded after transparent and waxdip, cell lump paraffin section is made, after HE dyeing, you can observed.Tissue after the collector can fix censorship sample carries out whole embedded sections, serial section, improves positive rate, overcomes the phenomenon that cell overlap accumulation, uneven distribution, structure are unclear in smear, there is provided more accurately information.
Description
Technical field
The invention belongs to basic medical research technical field, is related to a kind of device for collecting cell in humoral specimen, especially
It is related to a kind of cell mass collector；The invention further relates to a kind of method that cell in humoral specimen is collected with the collector.
Background technology
Traditional examination of castoff cells, the humoral specimens such as a small amount of chest, ascites, urine are exactly taken to carry out sample smear or centrifugation
Precipitation, takes a small amount of smear, therefrom searches tumour cell, has the advantages that method is simple, economic and pain is few and is answered by clinic
With.But its shortcoming is：After chest, ascites, urine centrifugation, its a little sediment is only taken to carry out smear, it is impossible to which sample is all made
Smear is observed, and is easily caused and is failed to pinpoint a disease in diagnosis and mistaken diagnosis, while cell smear is thicker, when observer lacks experience, easy mistaken diagnosis, particularly
It is easily caused false positive.
The content of the invention
It is an object of the invention to provide a kind of cell mass collector, can collect whole cells in a small amount of humoral specimen,
Do not easily cause and fail to pinpoint a disease in diagnosis and mistaken diagnosis.
Another object of the present invention provides a kind of method that cell in a small amount of humoral specimen is collected with above-mentioned collector.
To achieve the above object, the technical solution adopted in the present invention is：A kind of cell mass collector, including first from
Heart pipe, the support tube of tubular is provided with the first centrifuge tube, is provided with the second centrifuge tube in support tube, the tapered portion on the second centrifuge tube
Point stretched into through support tube in the conical section of the first centrifuge tube, the side wall of the second centrifuge tube conical section be provided with it is multiple go out liquid
Hole, in use, filter paper is put into the conical section of the second centrifuge tube, and filter paper is set to be bonded with the inwall of the conical section.
Another technical scheme of the present invention is：One kind collects humoral specimen using above-mentioned cell mass collector
The method of middle cell, specifically carry out according to the following steps：
Step 1：1 ︰ 9 by volume, formalin is added in humoral specimen, sample is fixed, after natural subsidence, discards
Supernatant, stay sediment；
Step 2：Sediment to be injected into centrifuge tube, centrifuged, abandoning supernatant, the drop of taking precipitate 1~2 carries out routine smear,
Ethanol is added in remaining sediment, after mixing, 1 drop protein chelate agent is added, mixes, obtain suspension；
Step 3：Suspension is filled on filter paper, centrifuged, the clear liquid in suspension passes through filter paper, is entered by fluid hole
In first centrifuge tube, the solid granule in suspension is trapped on filter paper, forms cell mass；
Step 4：Cell mass is embedded by existing method, be dehydrated, embedded after transparent and waxdip, cell block stone is made
Wax is cut into slices, after carrying out HE dyeing, you can observed.
Collecting cast-off cells making specimens paraffin embedding slices with collector of the present invention has advantages below：
1）Tissue after the cast-off cells such as the chest of censorship, ascites, urine sample being fixed carries out whole embedded sections,
And can serial section, reduction fails to pinpoint a disease in diagnosis, and improves the positive rate of cast-off cells.
2）Overcome the phenomenon that cell overlap accumulation, uneven distribution, structure are unclear in smear, there is provided more accurately information.
3）The specimens paraffin embedding slices of making can preserve for a long time, cut again repeatedly, and can do a variety of specific stains or SABC,
Classification to malignant tumour is very helpful, and reduces mistaken diagnosis, and foundation and conveniently is provided for clinical treatment.
4）Not by cytolgical examination is antifreeze, anti-corrosion, time length are limited, can stay at any time in the bottle with fixer,
The amount of leaving and taking of sample also can suitably increase.
Brief description of the drawings
Fig. 1 is the structural representation of collector of the present invention.
The microscope figure that hydrothorax cast-off cells are detected with existing cell smear method（HE is dyed, and 40 × 10）,
See Fig. 2 and Fig. 3.The microscope figure that the hydrothorax cast-off cells are detected with the inventive method（HE is dyed, and 10 × 10）, see
Fig. 4~Fig. 7.
The microscope figure that above-mentioned hydrothorax cast-off cells are detected with the inventive method（HE is dyed, and 10 × 10）, see figure
8~Figure 11.The microscope figure that the hydrothorax cast-off cells are detected with existing cell smear method（HE is dyed）, see figure
12~Figure 15.
In Fig. 1：1. the first centrifuge tube, 2. second centrifuge tubes, 3. support tubes, 4. fluid holes.
Embodiment
The present invention is described in detail with reference to the accompanying drawings and detailed description.
As shown in figure 1, collector of the present invention, including the first centrifuge tube 1, the 1 interior support tube for being provided with tubular of the first centrifuge tube
3, the lower end of support tube 3 is in contact with the cylindrical tube of the first centrifuge tube 1 and the joint portion of conical section, and the is provided with support tube 3
Two centrifuge tubes 2, the conical section on the second centrifuge tube 2 are stretched into the conical section of the first centrifuge tube 1 through support tube 3, and second
The side wall of the conical section of centrifuge tube 2 is provided with multiple fluid holes 4.
During using collector of the present invention, filter paper is put into the conical section of the second centrifuge tube 2, and make filter paper and the taper
Partial inwall fitting.
Present invention also offers a kind of method that cell in a small amount of humoral specimen is collected using above-mentioned collector, it is specific press with
Lower step is carried out：
Step 1：1 ︰ 9 by volume, 20~80mL of formalin of mass fraction 35~40% is added into 200~800mL bodies
In liquid sample, sample is fixed, then 30~60min of natural subsidence, abandoning supernatant, stay sediment；
Filter paper is put into the conical section of the second centrifuge tube 2, and filter paper is bonded with the inwall of the conical section；
Step 2：30~50mL of sediment is injected into centrifuge tube, 10min is centrifuged with 1500~2000r/min rotating speed, abandoned
Supernatant is removed, the drop of taking precipitate 1~2 carries out routine smear, the ethanol of 1mL mass fractions 95% is added in remaining sediment,
After mixing, 1 drop protein chelate agent is added, mixes, obtains suspension；
The protein chelate agent is egg, and dosage 1 is dripped, and about 50 μ L, its dosage can not be excessive, is otherwise influenceed in cell mass
Cell quantity.Egg white is denatured by ethanol, is frozen into lumps.
Step 3：Suspension is filled on the filter paper in the second centrifuge tube, centrifuged with 1500~2000r/min rotating speed
10min, the clear liquid in suspension pass through filter paper, are entered by fluid hole 4 in the first centrifuge tube 1, small of the solid in suspension
Grain is trapped on filter paper, forms cell mass；
Step 4：Cell mass is embedded by existing method, be dehydrated, embedded after transparent and waxdip, 4 μm of thickness is made
Cell lump paraffin section, carry out HE dyeing after, you can observed.
The centrifuge used in collection method of the present invention is consistent with centrifuge used in conventional cell smear.
In the present invention, using formalin to the timely fixation of sample, make cell protein denaturation in body fluid, keep cell former
There is form, prevent the further regression of cell, nuclear swelling, avoid being difficult to differentiate with tumour cell on morphology.Utilize albumen chela
Mixture makes to be scattered, single cell condensation into lumps, be easy to embed, cut into slices；And the protein chelate agent is in immunohistochemical staining
As a result in judging, no background colour developing.
Paired observation research is carried out to 75 samples：
1st, materials and methods
1.1. material：Choose hydrothorax to censorship in 09 month Baiyin City First People's Hospital in January, 2014, ascites sample 75
Example, all samples make cell mass using cell mass collector.
1.2. method：The sample of censorship, it is typically suitable with 200~800mL.By hydrothorax, ascites sample measurement volume, and press
Formalin is added in sample according to the ︰ 9 of volume ratio 1, makes sample with 10% formalin（Pure formalin concentration be 35~
40%, and the fixer of clinical conventional fixed preparation is 10% formalin, i.e.+9 parts of distilled water of 1 part of formalin）It is fixed.It is natural
30~60min is settled, abandoning supernatant, 30~50mL of sediment is stayed, inserts in 50mL centrifuge tubes, centrifuged with 2000r/min
10min.Abandoning supernatant, the drop of taking precipitate 1~2 carry out routine smear, and 1mL95% ethanol is added in remaining sediment,
Sediment is mixed, 1 drop egg is added, mixes；By mixed liquor be entirely within cell mass collect instrument in, with 2000r/min from
Heart 10min, directly takes out cell mass, is embedded, after conventional Full automatic specimen dewater treatment machine dehydration, transparent, waxdip
Embedding, is made 4 μm of thick cell lump paraffin sections, carries out HE dyeing, SABC detection CK, MC, Calretinin,
The expression of TTF-1, Napsin, CEA, Ki-67 immune marker, cell section and conventional cell smear paired observation, enter one
Step differentiates the good pernicious of Pleural effusions.
1.3. reagent：Immunohistochemical staining process monoclonal antibody:CK、MC、Calretinin、TTF-1、Napsin、
CEA, Ki-67 mouse anti-human monoclonal antibody and kit UltraSensitive SP step neoformation technological development purchased from Foochow to be had
Limit company is operated by kit specification, and PBS makees negative control instead of primary antibody.
1.4. result：Cytodiagnosis standard according to《Cell pathology diagnosis atlas and experimental technique》, Cao Yuehua etc. is main
Compile, Beijing science tech publishing house publishes.All smears need 2 experienced pathology associate chief physician diagosis；SABC contaminates
In color, CK, MC, Calretinin, Napsin, CEA protein expression in cell membrane, Ki-67, TTF-1 protein expression in nucleus,
It is in brown yellow granule;Positive cell>10% is the positive.
1.5. statistical procedures：Statistical analysis is carried out using SPSS17.0 statistical softwares, qualitative data uses χ2Examine.
, result
2.1. the laboratory counts to 2009~2014 years 1~September part same period chests, ascites sample cancer cell recall rate
Research, its result such as table 1.
Table 1 2009-2014 same periods chest, ascites sample cancer cell recall rate %
Statistical result showed, 1~September part same period pectoral ascites cancer cells Positive rate is 27%, 2014 within 2009~2013 years
Same period pectoral ascites cancer cells Positive rate is 67%, is conventional commonly more than 2 times of smear pectoral ascites cancer cells Positive rate,
The two has statistical significance（p<0.05）, its reason is analyzed, the cell production process of mainly common smear is lack of standardization, it is impossible to
To censorship chest, all cells of ascites carry out detection is key reason, and secondly, cell smear SABC effect does not have cell section
SABC effect is good.
2.2. 75 chests, ascites cast-off cells Immune expression feature（Table 2）.
2 75 chests of table, ascites cast-off cells Immune expression feature
Result of study is shown, in Malignant Pleural, cancer cell Ki-67 nuclear breeding indexes are 5%~70%, average left 35%
The right side, CEA positive rates 75%~95%, average 93%, CK positive rates 100%, Calretinin positive rates 10%, MC positive rates 16%,
TTF-1 positive rates 77%, NapsinA positive rates 67%, any of the above immunophenotype are mainly expressed in atypia cell；Good
Property hydrothorax in, Ki-67 nuclear breeding indexes are 1%~10%, average in 5% or so, CEA positive rates 5%~15%, average 11%, CK sun
Property rate 100%, Calretinin positive rates 96%, MC positive rates 88%, TTF-1 positive rates 4.4%, NapsinA positive rates 2.2%,
Any of the above immunophenotype is mainly expressed in the atypia cell for needing to differentiate with malignant cell, shows that the mesothelium of hyperplasia is thin
Born of the same parents.Ki-67, CEA, Calretinin, MC, TTF-1, NapsinA positive expression rate have aobvious in cancer cell and mesothelial cell
Write sex differernce（p<0.05）.In 30 cancer cell positive Pleural effusions, 25 visible malignant cells of hydrothorax, wherein gland cancer 15
Example, malignant mesothelioma 5, squamous cell carcinoma 2,1 t cell lymphoma, remaining 2 are neuroendocrine carcinoma.
Conclusion：Pleural effusions cast-off cells are collected using cell mass collector of the present invention, carry out pathological section, cell smear
Cellular morphology is observed, greatly improves pectoral ascites cancer cells recall rate；Cell mass collects instrument and collects Pleural effusions cast-off cells, enters
Row pathological section, is easy to immunohistochemical staining, and very big help is provided to staging.
Embodiment 1
1 ︰ 9 by volume, the formalin of mass fraction 35% is added in 200mL humoral specimens, fixes sample, so
Natural subsidence 30min afterwards, abandoning supernatant, stay sediment；Filter paper is put into the conical section of the second centrifuge tube, and makes filter paper
It is bonded with the inwall of the conical section；Sediment 30mL is injected into centrifuge tube, 10min is centrifuged with 2000r/min rotating speed, abandoned
Supernatant is removed, the drop of taking precipitate 1~2 carries out routine smear, the ethanol of 1mL mass fractions 95% is added in remaining sediment,
After mixing, 1 drop protein chelate agent is added, mixes, obtains suspension；Suspension is filled on the filter paper in the second centrifuge tube, with
1500r/min rotating speed centrifugation 10min, the clear liquid in suspension pass through filter paper, by fluid hole into the first centrifuge tube,
Solid granule in suspension is trapped on filter paper, forms cell mass；Cell mass is embedded by existing method, taken off
Embedded after water, transparent and waxdip, the cell lump paraffin section of 4 μm of thickness is made, after carrying out HE dyeing, you can observed.
The microscope figure that hydrothorax cast-off cells are detected with existing cell smear method（HE is dyed, and 40 × 10）,
See Fig. 2 and Fig. 3.The microscope figure that the hydrothorax cast-off cells are detected with the inventive method（HE is dyed, and 10 × 10）, see
Fig. 4~Fig. 7.As can be seen from Figures 2 and 3：A large amount of red blood cells, more amount lymphocytes and the single knurl of a little neutrophil leucocyte are thin
Born of the same parents, core is big, dye, caryoplasm deeply is out of proportion, and karyomorphism is irregular, and chromatin is deeper.Illustrate to be made with existing cell smear method
Smear it is thick, oncocyte be in single, no structure, is not easy and tumour cell difference.Fig. 4~Fig. 7 is shown：See adenoid, mamillary, mulberry
The oncocyte of Shen samples arrangement, core is big, dye, caryoplasm deeply is out of proportion, and karyomorphism is irregular, and chromatin is deeper.Illustrate thin with the present invention
The cell section for the cell mass that born of the same parents' collector is collected is relatively thin, and oncocyte arranges peculiar mode（Mulberries sample, mamillary, surrounding energy
There is complete line.
Embodiment 2
1 ︰ 9 by volume, the formalin of mass fraction 40% is added in 800mL humoral specimens, fixes sample, so
Natural subsidence 60min afterwards, abandoning supernatant, stay sediment；Filter paper is put into the conical section of the second centrifuge tube, and makes filter paper
It is bonded with the inwall of the conical section；Sediment 50mL is injected into centrifuge tube, 10min is centrifuged with 1500r/min rotating speed, abandoned
Supernatant is removed, the drop of taking precipitate 1~2 carries out routine smear, the ethanol of 1mL mass fractions 95% is added in remaining sediment,
After mixing, 1 drop egg is added, mixes, obtains suspension；Suspension is filled on the filter paper in the second centrifuge tube, with
2000r/min rotating speed centrifugation 10min, the clear liquid in suspension pass through filter paper, by fluid hole into the first centrifuge tube,
Solid granule in suspension is trapped on filter paper, forms cell mass；Cell mass is embedded by existing method, taken off
Embedded after water, transparent and waxdip, the cell lump paraffin section of 4 μm of thickness is made, after carrying out HE dyeing, you can observed.
The microscope figure that above-mentioned hydrothorax cast-off cells are detected with the inventive method（HE is dyed, and 10 × 10）, see figure
8~Figure 11.The microscope figure that the hydrothorax cast-off cells are detected with existing cell smear method（HE is dyed）, see figure
12~Figure 15.Fig. 8~Figure 11 shows, can see glandular tube, super bulk, the oncocyte of mulberries sample arrangement, and core is not of uniform size, pole is to disorderly
Disorderly, deep dye, caryoplasm are out of proportion, and karyomorphism is irregular, and chromatin is deeper.Illustrate the cell mass with cell harvester of the present invention
The cell section of block is relatively thin, and oncocyte arranges peculiar mode（Gland tubular arrangement, can around there is complete line）.Figure 12~Figure 15 shows
Show, a large amount of red blood cells, a small amount of lymphocyte and a little neutrophil leucocyte, part nucleus is big, dye, caryoplasm deeply is out of proportion, core
Shape is irregular, and chromatin is deeper, is arranged in tufted, mamillary, mulberries sample.The smear made from existing cell smear method
Thickness, oncocyte lumps or is dispersed in single, and eucaryotic cell structure is unclear, is not easy and tumour cell is distinguished.
Embodiment 3
1 ︰ 9 by volume, the formalin of mass fraction 38% is added in 500mL humoral specimens, fixes sample, so
Natural subsidence 45min afterwards, abandoning supernatant, stay sediment；Filter paper is put into the conical section of the second centrifuge tube, and makes filter paper
It is bonded with the inwall of the conical section；Sediment 40mL is injected into centrifuge tube, 10min is centrifuged with 1750r/min rotating speed, abandoned
Supernatant is removed, the drop of taking precipitate 1~2 carries out routine smear, the ethanol of 1mL mass fractions 95% is added in remaining sediment,
After mixing, 1 drop egg is added, mixes, obtains suspension；Suspension is filled on the filter paper in the second centrifuge tube, with
1750r/min rotating speed centrifugation 10min, the clear liquid in suspension pass through filter paper, by fluid hole into the first centrifuge tube,
Solid granule in suspension is trapped on filter paper, forms cell mass；Cell mass is embedded by existing method, taken off
Embedded after water, transparent and waxdip, the cell lump paraffin section of 4 μm of thickness is made, after carrying out HE dyeing, you can observed.
Claims (2)
- A kind of 1. method for collecting cell in humoral specimen, it is characterised in that the collection method is specifically carried out according to the following steps：Step 1：Cell mass collector is taken, the collector includes the first centrifuge tube（1）, the first centrifuge tube（1）It is interior to be provided with tubular Support tube（3）, support tube（3）Inside it is provided with the second centrifuge tube（2）, the second centrifuge tube（2）On conical section pass through support tube （3）Stretch into the first centrifuge tube（1）Conical section in, the second centrifuge tube（2）The side wall of conical section is provided with multiple fluid holes （4）, in use, filter paper is put into the second centrifuge tube（2）Conical section in, and paste the inwall of filter paper and the conical section Close；By volume 1：9, formalin is added in humoral specimen, sample is fixed, after natural subsidence, abandoning supernatant, stays First sediment；Step 2：First sediment is injected into centrifuge tube, centrifugation, abandoning supernatant, the second sediment is obtained, takes the second sediment 1 ~ 2 drops carry out routine smear, and ethanol is added in remaining second sediment, after mixing, add 1 drop protein chelate agent, mix, Obtain suspension；Step 3：Suspension is filled on filter paper, centrifuged, the clear liquid in suspension passes through filter paper, enters first by fluid hole In centrifuge tube, the solid granule in suspension is trapped on filter paper, forms cell mass；Step 4：Cell mass is embedded by existing method, be dehydrated, embedded after transparent and waxdip, cell block paraffin is made and cuts Piece, after carrying out HE dyeing, you can observed.
- 2. the method according to claim 1 for collecting cell in humoral specimen, it is characterised in that the egg in the step 2 White chelating agent is egg.
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| CN105547785B (en) * | 2016-01-14 | 2019-03-05 | 邵少慰 | A kind of preparation method of endometrium cast-off cells paraffin section and its application in Endometrial Carcinomas screening |
| CN107132088B (en) * | 2017-04-28 | 2020-07-17 | 上海市松江区中心医院 | Reagent for immediate coagulation of body fluid specimen sediment |
| CN116106108B (en) * | 2023-02-13 | 2024-04-05 | 杭州瑞普晨创科技有限公司 | Cell wax block and embedding method for frozen sections of small number of cells |
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