Biopsy - Science method

because this is a platform to discuss science and personal experience, we all can write on chatgpt, you clearly never used those things you are referring to, and for unknown reasons use chatgpt to give us pointeless answer. Please don't do it.

Hi Heather,

Can you share your isolation protocol ?

Thank you in advance,

Have you considered apoptosis or other cell death associated nuclear fragmentation?

Dear Rafal Z Slapa ,

Thyroid nodules are a common occurrence in the general population, and these incidental thyroid nodules are often referred for ultrasound (US) evaluation. US provides a safe and fast method of examination. It is sensitive for the detection of thyroid nodules, and suspicious features can be used to guide further investigation/management decisions. However, given the financial burden on the health service and unnecessary anxiety for patients, it is unrealistic to biopsy every thyroid nodule to confirm diagnosis.

Regards,

Shafagat

Hi Sasmita, I am culturing human foreskin fibroblast and facing a similar problem. After some time these thread like structures persist and later disintegrate into small floaty particles. How did you solve the issue. Please let me know?

Hi @Alberto Daniel Saucedo-Campose

I wan to focus on the inflammatory indexes and interaction of stroma cells and T&B cells.

Not relevant to to this discussion but somewhat related indirecly. When I was isolating mesenchymal stem cells from patients bone marrow taken from their femur undergoing knee surgery. There was variation among patients. Some patients' bone marrow stem cells did not grew further or very little grew. Whereas some patients bone marrow stem cells provided me millions of cells after 2 passages. I wanted to to go back to history of the patients what medicines and what diseases they had.

May be you should try to relate to patients medical history besides age group

The sensitivity of biliary brush border cytology is very low. A spyglass-bite biopsy under direct vision would be very useful. However, in the era of high resolution imaging, a proper triple phase MRI/CT should be able to diagnose Hilar CC. ERCP and Biopsy are not indicated as they may inflict an inflammatory reaction producing difficulty during surgery. Since Hilar CC can be treated with upfront surgery and does not require Neo-adjuvant CT, one may proceed with surgery after a radiological diagnosis supported by tumor markers.

Hello Giulia Lencioni

The correct procedure to cryopreserve human tissue samples would be as follows:

You may collect fresh tissue in sterile condition. You may cut small fragments (about 1-3 mm^3) and place them in cryopreservation medium in cryovials. The cryovials should be placed in isopropanol freezing container (cooling at the rate of -1 degree C /min) in -80 degree C freezer for 4 to 24 hours. Then transfer the cryovials in vapor phase liquid nitrogen for long-term storage.

You understood correctly that storing samples at -80°C is not recommended for long term storage. It is recommended only for a few months. As Georgy Leonov mentioned, transfer of human tissue samples from -80 degree C to -140 degree C should be done in less time to avoid thawing of the tissue samples. Try to keep the temperature as low as possible.

You could make use of dry ice. Depending on environmental conditions, you could use a cool box with crushed dry ice that will maintain the samples at -80 degree C. To extend the cooling duration, simply replenish the dry ice. Use of dry ice to transfer samples would help to avoid temperature rise and sample damage.

Best.

here are some general guidelines for bead selection:

Hello Miłosz Lewandowski

The skin samples obtained must be immediately frozen in liquid nitrogen and stored at −80°C until analysis. You may perform Western Blot within a month.

Best.

Take permission from the reputed hospitals and use their original data. you have to hide the patient's name by example like subject 1 Anirudh Gangadhar

Thank you everyone!

Thank you all for your kind response.

Where we can download the BCDR mammo database (especially the Digital part of the collection) ? No one seems to answer from the official site. Anyone who can provide a download link?

There is no absolute best, it can only be relative. As you see posts above (or even below) can be best in different situations, but you only you can decide what is best and practical for you.

>> Don't believe what ever you hear. Believe what you see in published research and read the specification / protocols of chemical you use or will you for anything. I think after that there should not be any fundamental related confusions.

From my perspective, freezing tissue biopsies -prior to metabolomics- in dry-ice could not replace freezing in liquid nitrogen. Because the aim of treating tissue with liquid nitrogen is to freeze samples quickly, which could avoid/reduce the damage of tissue. However, the freezing rate of dry-ice is obviously lower than liquid nitrogen. Thus, the use of dry-ice might not be a good idea.

As for the problems with transportation, I suggest that you may store the tissue in dry-ice after quick-freezing by liquid nitrogen.

I wish this answer could be helpful to you.

How did you do sample disruption and homogenization before RNA extraction?

Dear Paolo,

Thanks for your reply. At the moment GrowDex is for research use only but I'll let you know when GMP grade hydrogel is available.

There are many more learning approaches you could try. You may want to have a look at packages caret (https://topepo.github.io/caret/) or mlr3 (https://mlr3.mlr-org.com/)

Best Matthias

You may start by trying at first to calculate correlation coefficients (R) for both cases you are interested in and then to calculate possible regressions of your data, in order to analyze them. Furthermore, you may try to continue with ANOVA. Please also consult the paper attached which you may find helpfull for your analysis purposes

Dear Christian,

Theoretically, that should be possible. The (human?) blood samples and biopsies will also required higher safety standards. And under such high standards, cleaning and avoiding (cross) contaminations anyway need to be taken care of.

Needs to be carefully with the head of your institute, though.

Best,

MK

Hello Hanni Grace F

Autofluorescence can be a problem as it could interfere with detection of specific fluorescent signals, especially when the signal of interest is very dim. Most autofluorescence is detected at shorter light wavelengths with most absorbing in UV to Blue range (355-488 nm) and emitting in the Blue to Green range (350-550 nm). Autofluorescence can therefore be a problem in these light ranges as the signal to noise ratio is decreased resulting in reduced sensitivity and false positives.

There are several ways you could overcome autofluorescence.

1. As there is less autofluorescence at longer light wavelengths, fluorophores which emit above 600 nm will have less autofluorescence interference. The use of a very bright fluorophore will also reduce the impact of autofluorescence. So, choosing a fluorophore with emission spectra in the red and far-red regions will help distinguish specific staining from autofluorescence.

2. To lower tissue autofluorescence you can also treat the tissue with solutions of Sudan Black or similar non-fluorescent diazo dyes. These hydrophobic dye molecules will generally bind non-specifically to tissue sections. After binding to the tissue, Sudan Black acts as a mask to lower the fluorescence through the absorption of incident radiation (dark quenching).

3. Another method to diminish tissue autofluorescence is photobleaching. When this technique is used, tissue sections are exposed to high-intensity UV radiation for long periods of time to irreversibly photo-oxidize the fluorescent tissue elements. Photobleaching, which is often used in conjunction with other treatments, has been shown to be somewhat effective. However, it is time consuming.

4. You can also use the vector true view autofluorescence quenching kit which involves the treatment of tissue sections with an aqueous solution of a hydrophilic molecule that binds electrostatically to collagen, elastin, and RBCs. This non-fluorescent negatively charged molecule also binds effectively to formalin-fixed tissue including colon, pancreas, prostate, tonsil, spleen, kidney, gallbladder, and thymus.

Good Luck.

نعم يوجد

, do you have any specific company in mind regarding the biopsy punch? I tried 2 companies and they could not punch through. Have you used onefrom any specific company?

I think your answer is in the supplementary materials of this paper that you have cited https://static-content.springer.com/esm/art%3A10.1038%2Fncomms15824/MediaObjects/41467_2017_BFncomms15824_MOESM399_ESM.pdf on pages 25-26.

The authors have used the expression of hox genes from all the samples to cluster that data matrix and then used those clusters to create a new factor variable with x levels and used it as an adjustment factor in the regression model.

If you are asking how to create this cluster variable, then one way would be to use the 2 functions in R, you can google for a detailed usage

hclust and then cutree

You can also see a quick example from one of our project code

I don't know that I can give you a good sample size estimate for that. The most straightforward calculation, estimating sample size for detecting discordant scores in a sample, gives an n=1383 with a (assumed) prevalence of 10% at a 95% confidence level and a 5% margin of error with a 0.90 assay sensitivity. This would be quite the undertaking (>11,000 H&E stained slides to prepare and score).

I don't know that this is the proper calculation for your question, and perhaps I am misunderstanding what you are looking to do.

I would consider establishing an arbitrary, large sample size (e.g. n=100) and determining the proportion of discordant biopsies that you obtain. It's going to be difficult to determine biopsy sampling error without being able to analyze the entire uterus.

Some things to consider:

Easier to do it here https://www.medcalc.org/calc/diagnostic_test.php

Recently, TBSRTC, 2nd ed. has been using and Category III, atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS); IV, follicular neoplasm (FN) or suspicious for a follicular neoplasm (SFN); and V, suspicious for malignancy (SM) have been accepted as constituting indeterminate cytology of thyroid nodules. The risk of malignancies (ROMs) for indeterminate cytology are declared as 5-15%, 15-30%, and 60-75% in TBSRTC 1st ed. while 10-30%, 25-40%, and 50-75% in 2nd ed. for Category III, IV, and V, respectively, reflecting a wide range of ROM. The 2016 Royal College of Pathologists, United Kingdom (RCPath, UK) reported indeterminate cytology as Thy 3a, neoplasm possible, atypia/non-diagnostic and Thy 3f, neoplasm possible, suggesting FN, with the ROMs of 20-31% and 24-39%, respectively. In addition, the 2016 RCPath, UK declared Thy 4, SM with the ROM of 70-87%. The 2014 Italian Consensus for the Classification and Reporting of Thyroid Cytology (ICCRTC) divided diagnostic category TIR3, indeterminate cytology, into two subcategories, TIR3A (low-risk indeterminate lesion) and TIR3B (high-risk indeterminate lesion), with different expected ROMs and discrete clinical manners as <10% and 15-30%, respectively. The 2014 ICCRTC also harbors TIR4, SM with an expected ROM of 60-80%. The 2014 Royal College of Pathologists of Australasia (RCPA) and the Australian Society of Cytology (ASC) propounded: i) 3, Indeterminate (AUS/FLUS) with low ROM, 5-13%; ii) 4, Suggestive of an FN (FN/SFN) with moderate ROM, 21-26%; and iii) 5, SM with high ROM, 85-90%. The 2013 Japan Thyroid Association (JTA) Guideline for the Management of Thyroid Nodules declared: i) 3, Indeterminate, 3B, others; Indeterminate, ii) 3A, FNs, 3A-1, favor benign, 3A-2, borderline, 3A-3, favor malignant; iii) 4, Malignancy suspected, respectively. As such, each diagnostic category of the last TBSRTC, 2nd ed., with noninvasive follicular thyroid neoplasm with papillary-like nuclear features, NIFTP, harbor a wide range of implied ROMs.

I would suggest enzymatically digesting your minced tissue in a combination of two enzymes: type-1 collagenase (Worthington) and dispase (R&D Laboratories). Then plating the digested tissue in your complete medium on bare plastic for 12 hours at 37C. Telomerase negative fibroblasts should attach in that time frame.

Next, to induce proliferation of your fibroblasts, I would suggest you add PDGF-BB (R&D Laboratories) in base medium containing heat-inactivated serum (Atlas Biologicals, Fort Collins, CO). We used 10 ng/ml PDGF-BB for our telomerase positive stem cells in Opti-MEN + Glutamax (GIBCO) with 10% HI-FBS (Atlas Biologicals), pH 7.4 at 37C, so I suspect you will need to titrate the concentration of PDGF-BB that maximizes proliferation of your cells. Also remember, that as your cells increase in number you will need to feed your cultures more often if you expect survival.

The thing which is unclear in the question whether you are designing chip or trying to functionally use PDMS mould. I think these two are two different things.

Most of the times, leak is due to improper bonding which might be due to several reasons including improper/insufficient curing of the PDMS. Further, if you already speculate that your needles and tubing might be the cause, you could try to troubleshoot with it. In my experience, if the punch hole and needle are good fit, and the channels are clean before the bonding, there is no need of the resin.

you can github for search your relevant database

This might be helpful

Good luck

FAQ ID -2993 (Qiagen)

Lysis time varies depending on the tissue type processed. For recommended lysis times refer to the tissue protocol sheets. If lysis is incomplete after recommended lysis time, as indicated by the presence of insoluble material or highly viscous lysates, lysis time can be prolonged or insoluble material can be removed by centrifugation. Overnight lysis is possible and does not affect the preparation.

Hi,

In UK, MRD is done at following time points only:

1. At diagnosis (to identify the molecular marker)

2. Day 28 (end of induction chemotherapy)

3. Week 9 of treatment

4. Week 14 of treatment

Thank you everyone for you invaluable input. After intense discussion in my lab, I finally got an opportunity to run micro-array on my patient samples. I hope to find some answers soon.

Best,

Sakhila

Check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4016408/

Hello, Brazil does not have open databases for this type of data. I suggest looking for authors who have already published on this topic and have a database that you can share for your research.

Hi Lael,

You should be able to use a fairly standard method for starting primary fibroblasts. I wrote a detailed description of our method at RG a few years ago in the Questions asection. Look for it to get details. There are no enzymatic digestions, no specialized growth substrates, no cell type separations or centrifugations, or special growth media required.

We mince tissue biopsies into ~1 mm cubes using opposed sterile razor or scalpel blades, place the pieces into tissue culture grade plates without culture media, allow them to dry briefly (a few minutes) to promote adhesion, firmly press a dry sterile cover slip onto the biopsy pieces, add DMEM to the plate, and then culture for 10 days or so. The fibroblasts will grow out of the biopsy onto the coverslip and the culture plate. When they have grown out sufficiently, remove the cover slip, trypsinize the cover slips and the plate, and you’ve got primary Fb cultures. We used skin or other organs as the tissue source, but not intestine. However, the method ought to work on intestinal tissue too.

The method is effective due to the rapidity of Fb growth and division compared to other cell types, their strong adhesion to common charged substrates, and their resistance to damage by trypsin. They rapidly outgrow and out compete any other cell types present in the biopsy.

Good luck,

RW

Thank you for these suggestions.

With such a small biopsy, I would use IF microscopy on 1/2 of each biopsy to stain for cd3 and th1 v th2 v th17 cytokine signal co-localization.

I would then do qpcr on mrna extracts on the remainder of 3ach biopsy for all three cytokine families and tcr alpha beta and tcr gamma delta and then normalize the data.

Following

I imagine that your bicine and protein extraction won't damage your DNA from the biopsies. If your homogenization still leaves solid pieces that are in the pellet, they should be protected by shearing forces. Most DNA extractions start with a Proteinase K treatment anyway so doing a protein purification shouldn't hurt. We've also found that you may have to play around with your lysis buffer components to get at the pathogen genetic material in case you want to assay for the quantity of H.pylori in your biopsies. Our bacteria, T.pallidum needed SDS addition to get it away from the tissue and EDTA to stabilize the DNA.

It is not clear until you culture the suspected fungus. Ha ve you no culture capabilities?

Well!

There is direct and indirect involvement of the kidney by COVID 19 infection.

Acute kidney injury is a well known with proteinuria and rising creatinine.

There is still alot to understand by utilizing histological with various technique examinations.

It all depends on the resources but biopsy is always a good standard of care to understand what is going on with this new infection.

However, will it impact on patient care or not is the main question that must be addresed by the clinicians. Or is it just purely an academic aspect to further enhance our understanding of the pathophysiology mechanism.

Dear Conor Smith,

I agreed with Tsemay Tse even though it could help you to accelerate the keratinocyte culture but it depends on sample sources you got, sometimes it could affect your culture anyway. In our experience, you could use another technic by doing co-culture technique die to its eminent growth rate. However, you need to perform differential trypsinisation to ensure the removal of fibroblasts from keratinocytes. Best of luck!

put the tissue in freezing vials and Snap freeze in liquid nitrogen promptly then store -80.

Covadonga Vara

Hey Cova!

Which concentration were you using and which size of tissue?

Also do you do the lysis overnight or?

I have now tried lysing the tissue with PBS buffer and a bead before hand and it seems to work nicely.

Did you ever try this?

Hi Marianne,

I would check out IsoHelix Xtreme DNA isolation kits. The buffer used in the extraction keeps samples shelf stable for a while and might be a good alternative to RNAlater.

At least a few hundred to establish a reliable research. I think you have enough samples here.

Try to read this article it may help.

hi Murugadas A

Biswajoy suggested correctly about MACS. As we all know, MSCs are fibroblast cells, and identifying them from a particular source needs a rigorous step, MACS cell sorting is one of the best ways. Since such isolation consists of a heterogeneous population.

Well, come to your query, my understanding says

1. Since you have not mentioned your media type, I think MSCs grow nicely in KO/F12 media with glutamax.

2. Subsequent passaging will help you get rid of other cells.

3. MSC is of fibroblastic morphology, so subsequent passage will push or will gain more fibroblastic look as of now, in your condition, it seems only some cells have fibroblastic morphology i.e. tapering at the two ends. I am exactly not getting the group of cells thing as you mentioned, but many cases at initial stage cell cluster can be seen from where cells ooze outs.

4. Even if you sort the cells and get the MSC cell type, you will need to go for characterization. The MSCs proliferation is also necessary which you may see after the first passage since it's your freshly isolated and growing and adapting the initial in-vitro environment. Passage 2 to passage 4 will be right stage to characterize. Characterization can include population doubling assay, flow cytometry cell surface CD markers analysis, MSC markers gene, and protein expression. Vimentin immunofluorescence. {Note: Kindly check the intestinal tissue specificity}.

My answers are based on WJMSCs' isolation and characterization experience.

Regards

Saurabh

Search for a journal that suits your work

No way to use normal saline because such tissue spaceman underwent abnormal cellular & architectural changeswhich that may give a wrong results at diagnosis. Only use formalin & kept at room temperature.

Hello Jessica,

I would not come from experience side of these metabolomics sample matrices, but from robust literature of it!

1. (a) For Fecal Metabolomics, the best recent review that summarizes the methods and plus and minus of these are in Wishart's Review: https://www.sciencedirect.com/science/article/pii/S0003267018306354?via%3Dihub

(b) Plus, also looks like a good resource of direct comparison of fecal collection methods.

2. (a) For general metabolite stability, collection, handling and storage issues, I would suggest the BEST paper and esp all the Figures/ Boxes in there: if you read it in full, I do not think you will have more questions on sample collection, storage and handling BEST practice logistics for PLASMA, URINE, and other tissue samples as well.

and (b) Table 3 of this paper: https://www.ncbi.nlm.nih.gov/pubmed/31349624

and (c) of course the Fiehn's white paper is a great resource too: https://academic.oup.com/clinchem/article/64/8/1158/5608841

3. Urine Metabolomics: (new) and (old) could be two good starting points for your conclusions.

Hope the above helps and point you to right decisions for your samples!

Sorry no real answers but more possible readings!

Thanks,

Biswa

Hi Michal,

There is a filipin (compound similar to digitoxin) method for free cholesterol.

Fixation and sections: cryostat sections, post-fixed; frozen sections of fixed tissue.

Stock solution: 2.5 mg filipin in 1 ml dimethylformamide.

Staining solution: 0.2 ml stock solution in 10 ml PBS.

Method: wash sections in PBS.

Stain for 30 min in filipin solution.

Wash in PBS (x2).

Mount in PBS or glycerine jelly.

Examine by fluorescence microscopy (excitation BG12 with 512 nam barrier filter).

Results: Free cholesterol shows strong silvery fluorescence.

original method by Kruth & Vaughan 1980 - quoted in Bancroft & Stephens histology manual.

Good luck with your project.

It is a malignant tumor with long evolution and possibly with metastases. My option is to perform an amputation on the middle 1/3 of the leg.

not all bacterial may be see in slides , but in general we can see the bacterial colonies in infected tissue section, especially in severe bacterial infection

thank you for your answers everybody. Much appreciated

If that is the approach you are considering it will be wise to take both LBC sample and biopsy from each patient and conduct a comparative study. You may find that the two samples yield the same results. Hit two birds with one stone.

Hi Fahimeh,

You can do it. I did PCR genotyping of whole morula stage mouse embryos (8-16 cell stage) after doing the experiment. You can digest your cells in minimum volume of 10ul Proteinase K digestion solution in a small tube. You may cover the volume under paraffin oil (DNA-free oil, I mean, no source of contamination of any DNA source) just not to evaporate during incubation. Than follow your procedure of inactivation of proteinase K etc.

At the end, spin down, and use 1 ul inactivated Digestion mixture as DNA source (no need of isolation of DNA) for PCRing in a total of 10ul total reaction mixture (including water and master mix), cover under oil. Most of the time it worked. You can practice several times on discarded tissues before doing on real embryos. You may need good practice how to handle small volumes and avoid any DNA contamination.

I am enclosing reprint that applied to my gene only. You can use your PCR protocol and your PCR program and do not get confused my PCR program but get general idea that it is possible.

Good luck

Subhash

following

following

There is some research being done on urine as a liquid biopsy sample. If you are particularly interested in cancers associated with the urinary tract, perhaps this might be an area to explore.

As the tests are to be translated into a clinical environment additional steps like purifying RNA are not feasible, measurements have to be done on the protein extract, preferably by ELISA or WB. There are only tiny tissue samples available so measuring pH is also not an option. I looked into caspase cleavage products, but as the nerves are sick it is likely that there is disease-related apoptosis and also Wallerian degeneration that makes it difficult to use these processes as a linear marker.

The quality of the picture didnt allow to establish if it correspond to intestinal metaplasia. I sugest you to do another endoscopy with high definition and magnification.

The patient may have dihydropyrimidine dehydrogenase deficiency. That may cause decreased drug clearance and increased chance of toxicity.

Hi there.

So, regarding the surface markers to identify each of B cells & Treg cells (CD4+) populations, I recommend the following abs:

- For B cells, I recommend using mainly : CD19 and CD20. However, you can also add: CD27 (Memory), IgM & IgG. We also use IgD in flow cytometry but I'm not sure if it works with IHC or IF. Unless you purify your Bs & then stain them afterwards.

- For Tregs, you can use the following surface markers : CD3, CD4 & Foxp3.

Hope this gonna help. Good luck! :)

Hi Wolfgang Doerr

Thank you for your answer.

The source of the cell line we want to establish are from biopsys taken from patient. can you send me a link to a standard protocol?

thanks again!

The only way I see - as in almost all tissues apart from bone marrow there is no specific stem cell marker - the only way is to establish a cell culture procedure and look for clonogenic, i.e colony-forming cells. They may not reflect the true number of stem cells, but will allow for the estimation of relative changes.

W.

Dear Benedicta, in this moment we used breast biopsies fresh.

figured it out....

A simple 120 lbs recurve crossbow will do it, 150lbs max. You should find it in any fish/hunting stores, or online. No need for scope or other acessories, so it shouldn't cost more than 80-100 dollars. There are several inexpensive brands with similar looking crossbows (Buffalo River, Mankung Kantas, etc..). You may want to get an extra bow and strings to replace eventually, depending on how much you will use it. But you will have to buy the darts separately. You will need arrows with a float and biosy tips specifically designed for this (around 40mm length). Don't forget the permits for biopsying :). I hope this helps.

Was he on steroids for leukemia or a non malignant condition.? Probably the best way would be to wait till the peripheral blood total and differential count becomes normal or close to normal for non leukemic conditions.

Dear Renata Colombo Bonadio ,

Yes, i agree that anatomopathological diagnosis has not been replaced by imaging.As we move towards liquid biopsies and OCT , this may be the future.

Dear Dr. Pankaj

Thanks for sharing your thoughts and the reference which shows important for ruling out true negativity.

I do accept that most of the fastidious organism would fail to grow or even might due early history of antibiotics taken. But in this case we have seen gram negative bacteria from the vitreous by direct smear. Culture were negative. PCR targeting 16srRNA of eubacterial genome showed positive but the sequencing subsequently followed shoe no significant similarity. What could be the reason for the same. Though the organism is seen in direct smear and also PCR positive, why the sequence results not providing any evidence inspite of repeating the molecular tests with all sterile precaution.

Hello. You should consider the possibility that proteinase K (used for IDO) could affect the epitope of CD1c and CD141; and hence may affect their detection by the antibodies. In my opinion, it is better to perform antigen retrieval steps separately. Good luck.

For our studies, we used UV crosslinker from Stratagene (see link below). Practically, all cell lines (or primary cells) that are to be exposed to UV for crosslinking require some optimization for exposure. Most details for exposure of cells are provided in the manual of the crosslinker.

https://ipmb.sinica.edu.tw/microarray/index.files/Stratalinker%20UV%20Crosslinker.pdf