Histopathology - Science topic

Hello Moses Grimaldo Carjevschi

Histologically, granulocytic sarcoma (GS) undergoes various morphological changes in the cells. It is composed of immature cells of the neutrophil granulocytic series. The infiltration of immature, poorly differentiated cells with round to oval nuclei is the predominant finding. Crystalline, rod-like, intracytoplasmic acidophilic bodies called Auer rods are sometimes seen. With these histological findings, it is hard to distinguish GS from other lymphomas including histiocytic lymphoma, lymphoblastic lymphoma, Ewing's sarcoma, lymphocytic leukemia, undifferentiated carcinomas, and primitive neuroectodermal tumors.

You may refer to the article attached below.

Anti-lysozyme and chloroacetate esterase can be used to diagnose GS.

There are two types of esterases, specific and non-specific. The specific esterase will enzymatically hydrolyze naphthol AS-D chloroacetate liberating a free naphthol compound. This then couples with a diazonium compound, forming highly colored deposits at sites of enzyme activity. This enzyme is usually considered specific for cells of granulocytic lineage. This specific esterase stain, chloroacetate esterase (also called Leder stain), stains neutrophils, neutrophil precursors, eosinophils, and mast cells and confirms the granulocytic nature of the tumor.

On the other hand, the nonspecific esterase activity (alpha-napthyl acetate esterase) is seen in monocytes. The cells of granulocytic series are negative for the nonspecific esterase activity.

So, in the article regarding IHC used in myeloid sarcoma in which there was a report on a positive reaction to neutrophil esterase means that the author is referring to the specific esterase namely, chloroacetate esterase.

Best.

Factors such as concentration of the dye, pH of the staining solution, temperature, and tissue fixation are the most common factors that affects dye binding.

Mason's Trichrome stain is used to highlight connective tissue fibers in a tissue section.

Hi Monika,

If your specimens are medico-legal related, it should depend on your institutional law regarding the specimen ownership and disposal. Concerning specimen integrity, prolonged formalin storage will harden the tissues and cause formalin pigments (artifacts). Should you need the wet specimens in the future, I would advise changing the formalin solution once in a while.

Metaplasia

I think Ankush truth answer .

Hi

I think the eosinophilic material in Bowman's space is protein debris. I advise looking for signs of proteinuria such as hyaline casts. Indeed, occasionally within the renal tubules there appear to be small amounts of eosinophilic (proteinaceous) material...

If that is what you mean, I have only found information on effect on haematology and biochemistry and faecal loads (in broilers) and its antiviral effects.

From Royal Society of Biology RSB discussions

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:

Shreshtha Gaur….My colleagues use Adobe Photoshop for general purpose work. Those that do specialized image analysis use special software that came bundled with hardware or a variety of open source packages. The most well known and developed is "Image J" or one of the many custom and free variations (download here…https://imagej.nih.gov/ij/) - Image J also has an extensive on-line community that develops tutorials and plug-ins for specific fields of research and reading special file formats. You can read more here…

Another site that offers an overview of some other open source packages is here…

Until you receive other ideas I'd recommend two things; (1) starting with Image J and (2) ask the people at your university what they use. Having a nearby source of help is worth a lot.

Please avoid the Rome conference with everything you have. The journals are bad, very bad (even different journal names on the same PDF) and a google search for "is waset.org legit" has lots of reasons to avoid this completely. One headline on an investigation of them: "How the World Academy of Science, Engineering and Technology became a multimillion dollar organization promoting bullshit science through fake conferences and journals." I am not sure if Lotenna is paid by them to promote the conferences or is just naive, but please do NOT get yourself into that rat hole.

Most of the time, it is possible to know if muscle has been included in the specimen from visual inspection and chip thickness. If doubt remains, it is prudent to take biopsies from the floor with cold-cup forceps.

Image J software.

Movat's Pentachrome.

Pretty complicated.

T@@o provide baseline data on the gill tissue lesions in Huso huso under exposure to direct and water-soluble fraction of diesel oil. In both condition, histopathological analyses exhibited many gill lesions, including epithelial lifting, erythrocyte infiltration, lamellar aneurism, hyperplasia, and lamellar fusion. We report here the results on several abnormalities in the gill structure of the studied fish under direct and water-soluble fraction of diesel oil concentrations which could be used as biomarkers of diesel oil pollution.

Dear Anmol Banerjee ,

Previously I used a semiquantitative method for fat accumulation assessment in hepatocytes. the following sentences of our paper (2017) describe the method:" For each section, four to six unbiased counting frames were sampled in a systematic random fashion. Fat accumulation in the liver was graded as previously described (Brunt et al. 1999). Briefly, Grade 0, no fat was found; Grade 1, <33% (light); Grade 2, 33–66% (mild); and Grade 3, more than 66% of hepatocytes affected by fat droplets "

- Brunt E M, Janney C G, di Bisceglie A M, Neuschwander-Tetri B A and Bacon B R (1999) Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. American Journal of Gastroenterology 94 2467–2474.

Hope this will meet your concerns,

Mehran

sure, I'd like to see the pictures - philageis@aol.com

Thank you Dr Ogunlade Babatunde for your answer. It is much appreciated.

Regards.

Sunday

Hi Muhammad,

1. Have you placed the bone in fixative (10% Neutral Buffered Formalin or 4% PFA)?

2. Decal in 10% Formic acid

3. Tissue processor protocol for bone

I have a few questions to ask you before I give you specific details.

Please contact me: tina.vanmeter@gmail.com

Regards,

Tina

Train dataset not available in this chalange..

" No training set is provided, participants should use their own data to develop a method. "

Test data can be downloaded from the  Zenodo platform

Hi Aswathy M a

based on your q: " If H and E images are stained from different labs, training and testing with different datasets won't work?? And for hue similarity, can I do color normalization ? "

I would suggest you do colour normalistion to all images as hue would be different. mixed both dataset and divide into train and test so your algo will learn & train on wider variety of image compositions.

In my opinion it's OK to add histopathological features because the reader better understand what the case you are publishing, especially if your case really requires a diagnosis with histopathology

Good day,

Equipment of Histopathology and Cytology lab depends from volume of biopsies which is planning to be, the clinical departments etc. I can give you some advises about Histopathology equipment, in my country Histopathology and Cytology are different departments.

Best regards,

Dmitry

Depends on what are you looking for in a bone. Bone could be mineralized or demineralized, fractured or embedded. I bit more information is needed.

Dear Ahsan Mukhtar Rana,

Look the link, maybe useful.

Regards,

Shafagat

Dear Nattan,

If you want to show that the antibody specifically detects the viral proteins, an appropriate control would be lung tissue of an animal (or patient) not infected with that virus.

If you use the same staining protocol and microscope settings, you should not see staining in the control (not-infected) tissue, while the infected tissue should give a clear staining.

If this does not work put, you should reduce concentrations of the antibodies, use a different blocking solution (add horse serum, fish gelatine, etc), perform more/longer washing steps and, if the antibody still does not work properly, contact the company. Usually, you can get a different antibody for the same antigen for free, if you show that an antibody showd unspecific binding.

Best,

Sebastian

Epithelioid hemangioendothelioma?

Hello,

You can find the data in kaggle.com (has a lot of information about several topics) :

https://www.kaggle.com/paultimothymooney/breast-histopathology-images

and as a extra information here is a post to handle this data:

https://www.pyimagesearch.com/2019/02/18/breast-cancer-classification-with-keras-and-deep-learning/

I'm not a doctor, I am a biologist who worked on cancer previously, and sometimes work on cancer cells now. I was taught that p16 is BAD, and you should have a "heads up!!" reaction whenever you find it overexpressed (see the article). I would check the HPV positivity in the woman, possibly try to get genome data on her (for any unusual cancer-associated mutations, I'm not sure how much and what you can do in your setting) and generally monitor her in the future regularly. Because p16 is NOT GOOD, p16 means "think cancer". So I was taught, at least.

Thank you Melissa Chernick, Daniel Turkewitz, Emmanuel Ifeanyi Obeagu for your guidance,

There are similarities between IgG4RD and scleroderma. For example, T-lymphocytes seem to be of particular importance in both the diseases. These cells are predominantly CD4+ with a predominant Th2 cytokine profile characterized by high levels of IL-4, IL-5. This key role of T cell proliferation and cytokine secretion suggests that both these condition could be associated with a defective control of T cell activation. B cells are also activated and, through the production of autoantibodies, forced fibroblasts to switch in a profibrotic phenotype. Macrophages in perivascular infiltrates produced transforming growth factor beta that promote fibrosis. However, to diagnose an IgG4RD, histopathological hallmarks must be respected: 1) dense storiform fibrosis and 2) lymphoplasmacytic infiltrate rich in IgG4+ plasma cells (with a definite cut off of IgG4+/IgG+ ratio in the various tissues examined). The IgG4 antibodies in scleroderma are not the most representative and most expressed. In contrast to systemic sclerosis, in which the tissues fibrosis remain largely intractable to treatment, many IgG4-RD patients appear to have a condition in which the collagen deposition is reversible. Therefore, there are some similarities but also substantial histological and pathogenetic differences which must be investigated.

According to my experience in brain sectioning for special staining and brain topography .... you need brain slicer

if you need brain for frozen sectioning.

B-lymphocyte responses in the large intestine and mesenteric lymph nodes of mice infected with Eimeria falciformis (Apicomplexa). Nash PV et al. J Parasitol. (1988)

You can work with cytological specimens both retro- and prospective but (see the answer of Olena Polyakova) will need histological (surgical) material to make the diagnosis of NIFTP.

1. Defrost the hippocampus.

2. Immediately make an incision by the midline in the tissue with a scalpel.

3. Then put the sample in 10% formalin pH 7.2 to fix it for at least 2 hrs.

4. Perform the inclusion in paraffin and continue with the histopathology routine technique ...

Freeze a fresh, unfixed tissue sample, up to 2.0 cm in diameter, in OCT in a suitable tissue mold. Freeze the OCT containing the tissue onto the specialized metal grids that fit onto the cryostat.

Your question needs more elaboration. Variations in technique happens according to the need for specific methodology. In addition to Teresa GB's answer I just want to add 30% sucrose solution (for over night) for better sectioning results.

1.Sigma chemical company

2.Merck

Thanks, but I need the data for the images you need to process in MATLAB.

I think you could have some hair contaminating these tissue samples. The regular striations could be pigment bodies.

A very good question. Recurrent chalazion is a risk. However patients under immunotherapy ,recurrent ulcerative blepharitis and with other malignancy or known to harbour the risk should be kept a close eye on. And another practice that should be commissioned is -Every piece of tissue excised from the body mandates a histopathology with proper technique of biopsy.

There is no mechanism as such through which the nucleus avoids sectioning during microtomy. It is a matter of size of the nucleus of your cell sample and thickness of the section you fix during microtomy that decides how frequently you get sections of nucleus on slide. By the way are these paraffin embedded tissue sections? Which cell types form your sample?

I have done histology of ovaries and ovarian follicles of fish, frog and mouse. I get not only nucleus also nucleolus in histological sections. Also try serial sectioning and number them consecutively, I am sure you will not miss sections of nucleus.

In my opinion you have to look at the single constituents. It depends on the concentration which one is the main effector in the embalming fluid. If the ethanol is of high percentage (96-100%) you will get an hardening effect at the surface, lower penetration-rate. If you use lower concentrations it will rather act as a dilution for the formaldehyde with milder denaturation. Formaldehyde has an optimal ph-milieu for binding to specific functional groups of aminoacids. It may act better in aequous than in alcoholic solution. And the presence of ions can influence the pH, the binding-quality and -preference of formaldehyde etc.

A high ion-concentration may mask the binding-sites for formaldehyde, but may act for itself as dehydrating agent.

In older literature you find many fixative-mixtures, and each of it has a special aim. But I think, they were found by trial and error and the scientific reasons are not documentated well.

I am not clever enough to give a really good answer. - maybe someone else?

To see neuron/glia in hippocampus, Niissl stain (Cresyl voilet) is the conventional one.

Dr. Sorrentino absolutely right. Also depends of stage and area of burn injury.

I would recommend to check ICH S5(R2) guideline (http://www.ich.org/products/guidelines/safety/article/safety-guidelines.html) and the relevant OECD guideline, e.g. OECD 443 (https://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-4-health-effects_20745788?page=1).

Please be aware that you need detailed knowledge on testes/ovaries histomorphology in order to be able to do staging.

Hope that helps.

Best regards, Geertje

.14 days study is enough.it is possible to do histopathological test

Thank you so much for your kind reply. I appreciate your guidance and the list of papers and open sources.

Poupack

Dear Natheer,

Here are some resources:

The two articles I included are freely available online as well.

Hope this helps.

Best,

Kin

Hi

1. If the sample size of each group is >30, you can develop two independent T test.

2. Otherwise, non-parametric T test has to be developed.

3. For more information, please refer to:

. Landau, S. and Everitt, B. S.(2004). A Handbook of Statistical analyses using SPSS.

.Howitt, D. and Cramer, D.(2008). Introduction to SPSS.

Regards,

Zuhair

I'm not familiar with cardiac perfusion fixation so can you please help me on this regard by providing a authentic protocol ?

Honestly, just as a tip/pointer: Make sure the next time you are requested to make a diagnosis that the "Pathology Department in the Hospital" provides at least adequate (if not optimal!) material for evaluation [means: either reliably processed and stained original slides (tissue sections) or adequate images of the section(s), lesion(s') morphology (low mag,high mag), highlighting the most important guiding structures a pathologist MUST know to evaluate as well as to document / keep records of the found alterations, aiding and finally ending up in a correct diagnosis. And, naturally, without a separate formal request, there should be provided / added additional data regarding the patient's anamnesis, the clinical history / disease course, as well as additional compiled laboratory values. And therefore: I for 100% second the reply #005 as stated by Alexander Shtabsky....But: Thank you for letting the colleagues know what the diagnosis was....

I think this case is a diffuse form of renal amyloidosis

For histopathological evaluation in our study, the largest lobe of the liver was fixed in 10% neutral buffer formalin, embedded in paraffin and 5micrometer thick section was used for H&E staining. The scanned images were analyzed and gross morphological alterations could be seen. For an unbiased scoring, the identity of slides were blinded and evaluated (by pathologist) for histopathological lesions (necrosis, fat infiltration, fibrosis and mononuclear cell infiltration) and given a score that ranged between 0 (<5%) to 4 (>60%). The lesions observed fell under following categories viz none (score=0), minimal (score=1), mild (score=2), moderate (score=3) and marked (score=4). Further, the distribution of these lesions were also classified as focal, multifocal and diffuse.

Hope this may provide some help!

Agree with Dr. Aboumosalam,. In addition, if you worked on small animals like rats and mice, you can take LS of the kidney, this section give you a clear vision about the cortex and the medulla together so, you can detect any pathological features easily. In the liver I prefer to take a piece from each lobe to be sure that the pathological effect is localised or generalized.

Please share your experience if problem solved with above suggestions or not

You could prepare cell pellets on histological glass slides by cyto-centrifugation. Following centrifugation, cell pellets on slide could be easily fixed in ice-cold alcohol or a mixture of alcohol and acetone (50/50, v/v) for 5 min. Subsequently, the cell pellets could be immunocytochemically stained with antibody to cell proliferation-associated proteins ( i.e., ki-67 or topoisomerase II alpha). The later antibody is more specific to cell proliferation stage, where as the former recognizes cell in their cycling phase.

I am already very deep in literature survey since I am in my 3rd year of PhD. I am currently reading about field cancerization... And since my work focuses on histopathological analysis of images.. I couldn't find any good reference paper with respect to histological changes due to field effect. Hence thought to ask here. Thank you for your suggestion.

Try wrapping the heart in cling-wrap once extracted from the mouse and put it in a container and put straight into dry ice. Use 1% TTC in PBS, incubate the sections of the heart on each side for 10 mins at 37C. After 10 mins flip the sections and incubate again for 10mins. Then put the sections in 10% buffered formation and leave overnight at room temperature. That way you get the best contrast. Without formalin, the infarct area is hard to see.

Patient recieved adjuvant chemotherapy and is on surveillance more than 2 years, disease free. No rerescection required

she underwent flexible sigmoidoscopy every 6 months

I can tell you that early diagnosis of cutaneous melanoma on acral skin should rely on clinical pathological correlation. Early lentiginous melanoma on acral sites can be clinically very obvious but histopathologically very subtle. There are a some articles published regarding this issue. At least on volar skin, yes, melanoma can be diagnosed earlier on clinical grounds.

Arch Pathol Lab Med. 2011 Jul;135(7):847-52. Acral junctional nevus versus acral lentiginous melanoma in situ: a differential diagnosis that should be based on clinicopathologic correlation. Bravo Puccio F1, Chian C.

Am J Dermatopathol. 2014 Feb;36(2):142-7. Acral lentiginous melanoma: indolent subtype with long radial growth phase. Kim JY1, Choi M, Jo SJ, Min HS, Cho KH.

Ann Dermatol. 2011 Aug;23(3):400-4. Acral Lentiginous Melanoma Developing during Long-standing Atypical Melanosis: Usefulness of Dermoscopy for Detection of Early Acral Melanoma. Oh TS1, Bae EJ, Ro KW, Seo SH, Son SW, Kim IH.

QuPath Open source software for digital pathology

Orbit Image Analysis

Both software are easier than matlab. There cut images into tiles automatically.

Good day, dear Jun Wang 

Fig1 - it is too low magnification to understand what is situated in left part of figure, but right part is area of calcinosis

Fig2- area of necrosis and blood imbebition

Fig3 - Necrosis and particulary sclerosis of pseudocapsule

Fig4- Pseudocapsule of clearcell renal cell carcinoma

Fig5 and Fig6 - Areas of renal tisue with necrosis, so you couldn`t see any epitelium of tubules and endothelium of vessels. Due to cutting of thin sections there isn`t glomerulus.

Thanks Dibbanti and Maria for your valuable suggestions!

If your tissue is fixed with 4% paraformaldehyde , the best thing is to do 30/40 µm floating sections. and perform IHC in culture wells. After DAB, you spread them on super-frost+ glass slides and allow them to dry for 1 night before dehydratation and coverslipping.

With these thick sections, you see better the arborization of astrocytes.

thanks a lot

Yes, dear Dr

This important link

Thank you very much

I want to compare with wild type picture :)

Anyway, it looks like medial hyperplasia (between elastin lamina). 

Trichrome stain is helpful to detect elastin lamina.

Thank you so much William for your detailed explanation!!

@Adejoke: Do you mean the weight of the microtome? I only found the lightest can cut 5 micron is about 14 kg. This is considered ok. 

@Nabil: I want to find a "portable" microtome for field pathology. Hence, IHC may not be possible.

i agree it might be the secondary, have you tried using donkey anti-goat and donkey anti-rabbit? and block with donkey serum if you are using serum. Donkey anti-goat and donkey anti-rabbit secondaries can be found by lifetechnologies (thermofisher)