CN103994913A - Fixed-decalcifying fluid for bone marrow biopsy and paraffin section method of bone marrow biopsy tissue - Google Patents
Fixed-decalcifying fluid for bone marrow biopsy and paraffin section method of bone marrow biopsy tissue Download PDFInfo
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- CN103994913A CN103994913A CN201410065474.4A CN201410065474A CN103994913A CN 103994913 A CN103994913 A CN 103994913A CN 201410065474 A CN201410065474 A CN 201410065474A CN 103994913 A CN103994913 A CN 103994913A
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Abstract
The present invention provides a fixed-decalcifying fluid for bone marrow biopsy and a paraffin section method of bone marrow biopsy tissue. The fixed-decalcifying fluid includes 40% of formaldehyde, EDTA and a buffer solution, and has a pH value of 7.0-7.2. The reagent and the method provided by the invention merge and complete the two steps of bone marrow tissue fixation and decalcification at the same time, don not require liquid change in the middle, reduce the operation steps, shorten the process of specimen pretreatment, and are more conducive to giving diagnosis conclusion and treatment by the doctor. The obtained HE section shows well preserved cell morphology and clear tissue structure; immunohistochemistry staining shows excellent staining effect, accurate positioning and clean background; and the sample does not show the phenomenon of excessive decalcification.
Description
Technical field
The present invention relates to fixing-decalcifying Fluid and bone marrow biopsy tissue paraffin section de method for bone marrow biopsy.
Background technology
Marrow biopsy art is called for short bone marrow biopsy, makes pathological examination exactly by the cylindrical bone myeloid tissue that a special puncture needle is got the about 1.0-2.0 centimeter length of a fritter.Method of operating is identical with bone marrow smear centesis, and bone marrow biopsy has kept complete myeloid tissue structure, can reflect well myelosis degree, understands more all sidedly the bone marrow infiltration of marrow morbid state feature, especially extramedullary tumor.
The now normal paraffin section fabrication techniques bone marrow biopsy tissue that adopts, mainly comprises the following steps: the fixing of sample of bone marrow tissue, decalcification, dehydration, embedding, section, HE dye and need to carry out immunohistochemical staining by medical diagnosis on disease.The quality that affects marrow paraffin section is many-sided, and wherein the key link such as fixing, the decalcification of myeloid tissue, dyeing is dealt with improperly, all can affect the quality of section, thus affect doctor to pathological diagnosis result judge.
Making the most important link of high-quality Sections of Bone Marrow is the fixing and decalcification of sample tissue.Fully do not fixed if marrow sample is organized, in follow-up link, in sample tissue morphology structure and cell, various compositions will be destroyed so.If sample organizes decalcification incomplete, cannot cut out complete paraffin section, and easily fall sheet in dyeing course, easily cause artificially institutional framework imperfect.
Conventional marrow sample immobile liquid has 10% neutral formalin, Bouin liquid or LowyFMA liquid now.Conventional decalcifying Fluid has the gentle decalcifying Fluid such as mineral acid or EDTA.The neutral formalin regular time of use 10% is 16-24 hour, because if the set time is too short, may cause inhomogeneous immune response, and outmoded tissue is easier to chromogenesis precipitation.Bouin liquid comprises the glacial acetic acid with intense stimulus smell, and checker is in the time using this class immobile liquid, and respiratory system can be stimulated consumingly.In LowyFMA liquid, comprise the picric acid that belongs to explosive reagent, when operation, be very careful, use this class immobile liquid to have certain potential safety hazard.Use Bouin liquid and LowyFMA liquid fixed preparation tissue, also need 16-24 hour action time.
Conventional decalcifying Fluid has two classes now: one is mineral acid, the decalcification speed of this class decalcifying Fluid, and general bone marrow biopsy is organized decalcification 2 hours, but may cause the loss of many antigens, and immunohistochemical staining is had a significant effect.Another kind is gentle decalcifying Fluid, and as EDTA etc., but decalcification speed is slow, need to spend the night.
In existing bone marrow biopsy paraffin section method the fixing and decalcification link of sample tissue separately two steps carry out, first with immobile liquid, marrow sample is fixed, then immobile liquid is replaced with decalcifying Fluid and completes sample decalcification.What complete at ambient temperature sample with immobile liquid fixedly needs 16-24 hour, and the decalcification that completes sample with decalcifying Fluid needs 18-24 hour.So, whole fixing with decalcification approximately need to spend 24-48 hour.And so long sample of bone marrow microsection manufacture process directly causes providing the time increase that detects diagnostic result, the promptness of impact treatment.Moreover, in existing dicing method, fixing with decalcification is two steps separately, and each step needs the strict control time, once the inaccurate effect that will directly have influence on follow-up paraffin section of processing time of fixing or decalcification, therefore checker must find time and change working fluid (being replaced by decalcifying Fluid from immobile liquid) in checkout procedure, and such workload is very large.
On the other hand, for promotion health service business, the inspection business item that personnel, outfit are had relatively high expectations is given third party's independence medical laboratory has become trend.Like this sample that is dispersed in various big hospital is focused on to independent medical laboratory and complete various Interventions Requested.Hospital collects patient's sample and is transferred to third party's independence medical laboratory, passes on 1 day at least time, and 2-3 days at most.Existing bone marrow biopsy method need to be changed working fluid midway, and therefore existing method is just not too applicable to adopting third party's independent experiment chamber to complete bone marrow biopsy project.
Summary of the invention
In order to overcome the deficiencies in the prior art, the object of the present invention is to provide a kind of fix-decalcifying Fluid of the sample for bone marrow biopsy, comprise 40% formaldehyde, EDTA and damping fluid, pH value is 7.0-7.2.
Further, described damping fluid is phosphate buffer.
Further, in 1000ml, comprising:
0.1M phosphate buffer 900ml;
40% formaldehyde 100ml;
150 grams of EDTA;
PH value is 7.0-7.2.
The present invention also provides a kind of method of bone marrow biopsy tissue paraffin section de, comprise the following steps: the fixing-decalcifying Fluid of sample of bone marrow is fixed and decalcification, flowing water flushing, dehydration, waxdip, embedding, section, HE dyeing and/or immunohistochemical staining, it is characterized in that, described fix-decalcifying Fluid comprises 40% formaldehyde, EDTA and damping fluid, and pH value is 7.0-7.2.
Further, in 1000ml, comprising:
0.1M phosphate buffer 900ml;
40% formaldehyde 100ml;
150 grams of EDTA;
PH value is 7.0-7.2.
Further, sample: the volume ratio of fixing-decalcifying Fluid is equal to or greater than 1:10.
Further, the time of fixing-decalcification is 20-72 hour.
Further, the method for HE dyeing comprises: dewaxing, dyeing, dehydration, transparent and sealing.
Further, staining procedure comprises puts into haematoxylin dyestuff 6 minutes successively, washes 1 minute, and the differentiation of 0.5-1% hydrochloride alcohol for a moment, makes color be reddened by indigo plant, and tap water rinses approximately 10 minutes, and color is blue by red stain, and eosin stain is contaminated.
Further, tap water rinses 10 minutes colors after redness becomes blueness, with 95% alcohol processing.
The invention has the beneficial effects as follows: adopt of the present invention fixing-decalcifying Fluid and bone marrow biopsy tissue paraffin section de, two steps fixing and decalcification of myeloid tissue can be combined simultaneously and complete, in bone marrow biopsy tissue paraffin section de process, just do not need to change liquid midway, reduced operation link.Can at least shift to an earlier date 1 day than prior art reagent of the present invention and method and complete paraffin section, shorten sample pretreatment process, more be conducive to doctor and draw diagnosis, provide therapeutic scheme.The HE section showed cell form that adopts reagent of the present invention and method to obtain is intact, and institutional framework is clear; Immunohistochemical staining shows that Color is good, accurate positioning, clean background; Even sample is immersed in fixing-decalcifying Fluid for a long time, for example, through 72 hours, still there is not the phenomenon that decalcification is excessive in sample, and HE section and immunohistochemical staining are all uninfluenced.
Brief description of the drawings
Fig. 1 is case 1 bone marrow biopsy HE dyeing, shows that myelosis is active, the hyperplasia that volume is less than normal, but be difficult to accurately differentiate the cell derived of its hyperplasia.
Fig. 2 is case 1 CD3 immunohistochemical staining, shows the T lymphocyte in myeloid tissue.
Fig. 3 is case 1 CD20 immunohistochemical staining, shows the bone-marrow-derived lymphocyte in myeloid tissue.
Fig. 4 is case 1 CD79a immunohistochemical staining, shows the bone-marrow-derived lymphocyte in myeloid tissue.
Fig. 5 is case 1 CD235a immunohistochemical staining, shows the erythroid progenitor in myeloid tissue.
Fig. 6 is case 1 Lysozyme immunohistochemical staining, shows monocyte and granulocyte in myeloid tissue.
Fig. 7 is case 1 MPO immunohistochemical staining, shows monocyte and granulocyte in myeloid tissue.
Fig. 8 is case 1 PAX5 immunohistochemical staining, shows the bone-marrow-derived lymphocyte in myeloid tissue.
Fig. 9 is case 2 bone marrow biopsy HE dyeing, shows that myelosis is low, and the cell of differentiation and maturation is on the low side, but can not accurately identify initial cell wherein and assess its ratio.
Figure 10 is case 2 bone marrow biopsy HE dyeing, shows that myelosis is low, and the cell of differentiation and maturation is on the low side, but can not accurately identify initial cell wherein and assess its ratio.
Figure 11 is case 2 CD34 immunohistochemical stainings, shows initial cell and distribution situation thereof in myeloid tissue.
Figure 12 is case 3 bone marrow biopsy HE dyeing, shows that in marrow, hyperplasia degree is normal, reactive high plasmocyte, but can not accurate evaluation thick liquid cell ratio wherein judge whether it is monoclonal hyperplasia.
Figure 13 is case 3 CD138 immunohistochemical stainings, shows marrow mesoplasmatocyte hyperplasia and distribution situation.
Figure 14 is case 3 Kappa dyeing, shows thick liquid cell clone property hyperplasia situation.
Figure 15 is case 3 Lambda dyeing, shows thick liquid cell clone property hyperplasia situation.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.
The preparation of fix-decalcifying Fluid of embodiment 1 bone marrow biopsy tissue
In 1000ml, comprise
0.1M phosphate buffer (PBS) 900ml;
40% formaldehyde 100ml;
150 grams of EDTA;
Regulate pH value to 7.0-7.2.
Wherein, damping fluid, except PBS, also can be selected other applicable damping fluids.
The method of fix-decalcification of embodiment 2 bone marrow biopsy tissues
Bone marrow biopsy specimen is fixed and decalcification with the fix-decalcifying Fluid described in embodiment 1, wherein sample: the volume ratio of fixing-decalcifying Fluid is equal to or greater than 1:10.Fix-decalcification of normal temperature 20-72 hour, rinses with flowing water afterwards.Fixing and the decalcification of myeloid tissue of the present invention completes in fixing-decalcifying Fluid simultaneously.
The method of embodiment 3 bone marrow biopsy tissue paraffin section des
For the sample of bone marrow biopsy described in embodiment 1, example 2 fixing-decalcifying Fluid and method be fixed and decalcification 20-72 hour, flowing water flushing, dehydration, waxdip, embedding, section (slice thickness 2-3 μ M), HE dyeing and/or immunohistochemical staining, micro-Microscopic observation.Gained section can be directly used in microscopic examination, pathological examination, also can need to carry out immunohistochemical staining according to different.Gained HE biopsy tissues cell is intact, and Color is good.The positive accurate positioning of immunohistochemical staining, background is painted shallow.
Wherein the method for dehydration comprises: the myeloid tissue that has completed fixing decalcification processing is put into formaldehyde 60 minutes successively, water 10 minutes, 75% ethanol 60 minutes, 85% ethanol 60 minutes, 95% ethanol 60 minutes, 95% ethanol 90 minutes, 100% ethanol 60 minutes, 100% ethanol 90 minutes, dimethylbenzene 30 minutes, dimethylbenzene 40 minutes.
Wherein paraffin-embedded method comprises: the myeloid tissue that completes processed is put into paraffin 60 minutes successively, paraffin 90 minutes, paraffin 90 minutes.
Wherein the method for HE dyeing comprises: will complete after paraffin embedding, and section, and complete successively dewaxing, and dyeing, dehydration, transparent, sealing.
Wherein the step of dewaxing comprises: put into successively dimethylbenzene 5 minutes, and twice, 100% alcohol 1 minute, 100% alcohol 1 minute, 95% alcohol 1 minute, 80% alcohol 1 minute, tap water rinses 1 minute.
Wherein the step of dyeing comprises: put into successively haematoxylin dyestuff 6 minutes, wash 1 minute, the differentiation of 0.5-1% hydrochloride alcohol for a moment, makes color be reddened by indigo plant, and tap water rinses approximately 10 minutes, and color is blue by red stain, and eosin stain is contaminated.In preferred scheme, tap water rinses 10 minutes colors after redness becomes blueness, process for a moment with 95% alcohol, to avoid with moisture content diluted eosin stain, affect Color.
The step of transparently sealing of wherein dewatering comprises: put into successively 75% alcohol 0.5 minute, 85% alcohol I 1 minute, 95% alcohol II 1 minute, 100% alcohol I 1 minute, 100% alcohol II 1 minute, carbolic acid: dimethylbenzene is 10 seconds of 1:3 solution, transparent 1 minute of dimethylbenzene I, transparent 1 minute of dimethylbenzene II, uses neutral gum sealing after taking out.
Embodiment 4 utilizes the present invention to carry out bone marrow biopsy to clinical sample
Get 3 original sample of bone marrow of case, all utilize of the present invention fixing-decalcifying Fluid and method, be fixed-decalcifying Fluid is respectively 10ml, 15ml, 15ml, and the processing time is respectively 20 hours, 48 hours and 72 hours.Adopt the reagent of embodiment 1-3 and method to complete fixing, decalcification, dehydration, waxdip, paraffin embedding, HE section and/or the immunohistochemical staining of sample.Microscopic examination the results are shown in Figure 1-15.
In case 1:HE section, show the volume cell-nest less than normal that marrow medium mess shape distributes, according to HE, section can not go out respectively lymphocyte, erythroid progenitor or volume initial cell less than normal, through waiting immunohistochemical staining, micro-Microscopic observation, rake celluar localization is accurate, clean background, assisted diagnosis doctor accurately identifies cell derived.Fig. 1 is the HE coloration result of case 1, and Fig. 2-8 are respectively CD3, CD20, CD79a, CD235a, Lysozyme, MPO, the PAX5 immunohistochemical staining results of case 1.Bone marrow biopsy diagnostic result case 1 being completed by the inventive method is consistent with the diagnostic result completing with additive method.
Case 2:HE section shows that in marrow, cell concentration calibration ordinary person is on the low side, more visible volumes cell less than normal, is difficult to identify its initial cell ratio in HE section, through CD34 immunohistochemical staining, micro-Microscopic observation, assisted diagnosis doctor accurately identifies initial cell ratio and characteristic distributions.Fig. 9 and 10 is HE coloration results, and Figure 11 is CD34 immunohistochemical staining result.Bone marrow biopsy diagnostic result case 2 being completed by the inventive method is consistent with the diagnostic result completing with additive method.
Case 3:HE section shows that in marrow, cell concentration is roughly normal, visible thick liquid cell and slurry like cell ratio increase, but can not judge whether its thick liquid cell is monoclonicity hyperplasia in HE section, through CD138, Kappa, Lambda immunohistochemical staining, micro-Microscopic observation, can accurately identify plasmacytic ratio and distribution situation, and judge whether it is Kappa or Lambda monoclonal hyperplasia.Figure 12 is HE dyeing, and Figure 13 is CD138 immunohistochemical staining, and Figure 14 is Kappa immunohistochemical staining, and Figure 15 is Lambda immunohistochemical staining.Bone marrow biopsy diagnostic result case 3 being completed by the inventive method is consistent with the diagnostic result completing with additive method.
Can find out and utilize reagent of the present invention and method to process sample from the detection of above-mentioned case, shorten the processing time in early stage, through paraffin section, HE and immunohistochemical staining, micro-Microscopic observation: 1, HE section showed cell form is intact, and institutional framework is clear.2, immunohistochemical staining shows that Color is good, positive accurate positioning, clean background, the foundation providing for pathological diagnosis.On the other hand, in the time processing type specimen, do not need to change liquid, and can shorten fixing-approximately 24 hours decalcification processing times.Through the sample of the inventive method processing and the sample comparison obtaining by the method for existing routine, the present invention can reach the effect of existing conventional method completely, can meet the requirement of clinical examination diagnosis, and compare existing conventional method and greatly shorten detection time, operation steps is simple.And the phenomenon that decalcification is excessive does not appear in the sample that utilizes method of the present invention to process through 72 hours, HE section and immunohistochemical staining are all uninfluenced, the requirement of can satisfy the demand long period transport, depositing sample.And by existing conventional method through the sample of decalcification processing in 48 hours in contrast, result shows and there will be the excessive phenomenon of decalcification after 48 hours with existing conventional method decalcification processing, affect film-making result.
Claims (10)
1. for a fix-decalcifying Fluid of the sample of bone marrow biopsy, comprise 40% formaldehyde, EDTA and damping fluid, pH value is 7.0-7.2.
2. fix-decalcifying Fluid according to claim 1, is characterized in that, described damping fluid is phosphate buffer.
3. fix-decalcifying Fluid according to claim 1, is characterized in that, in 1000ml, comprising:
0.1M phosphate buffer 900ml;
40% formaldehyde 100ml;
150 grams of EDTA;
PH value is 7.0-7.2.
4. the method for a bone marrow biopsy tissue paraffin section de, comprise the following steps: the fixing-decalcifying Fluid of sample of bone marrow is fixed and decalcification, flowing water flushing, dehydration, waxdip, embedding, section, HE dyeing and/or immunohistochemical staining, it is characterized in that, described fix-decalcifying Fluid comprises 40% formaldehyde, EDTA and damping fluid, and pH value is 7.0-7.2.
5. method according to claim 4, is characterized in that, in 1000ml, comprising:
0.1M phosphate buffer 900ml;
40% formaldehyde 100ml;
150 grams of EDTA;
PH value is 7.0-7.2.
6. method according to claim 4, is characterized in that, sample: the volume ratio of fixing-decalcifying Fluid is equal to or greater than 1:10.
7. method according to claim 4, is characterized in that, the time of fixing-decalcification is 20-72 hour.
8. method according to claim 4, is characterized in that, the method for HE dyeing comprises: dewaxing, dyeing, dehydration, transparent and sealing.
9. method according to claim 8, is characterized in that, staining procedure comprises puts into haematoxylin dyestuff 6 minutes successively, wash 1 minute, the differentiation of 0.5-1% hydrochloride alcohol for a moment, makes color be reddened by indigo plant, tap water rinses approximately 10 minutes, and color is blue by red stain, and eosin stain is contaminated.
10. method according to claim 9, is characterized in that, tap water rinses 10 minutes colors after redness becomes blueness, with 95% alcohol processing.
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| CN110618010A (en) * | 2019-09-26 | 2019-12-27 | 江苏护理职业学院 | Method for manufacturing paraffin section of bone tissue |
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| CN116380589A (en) * | 2023-04-07 | 2023-07-04 | 武汉益特医疗技术咨询有限公司 | Pretreatment method and application of bone marrow tissue |
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Application publication date: 20140820 |