Thank you both very much for your thorough answers to my question; you have both given me a lot to think about and I am truly grateful. I think the overall take home message is to become good friends with a pathologist who has a expert eye for the subtle changes in tissue that can be overlooked by a novice.

H & E is simple cheap and quick but needs experience.Cells like neutrophils, plasma cells, monocytes, eosinophils and basophils are easy to identify.Lymphocytes are also identifiable and sometimes reacrive T lymphocytes can easily be differentiated from the smaller B lymphocytes. Most important is the quality of the staining and the section..

Sometimes we stain sections by Giemsa stain to look for leismania parasites and it gives a nice stain to these white cells But, I am afraid, if you looking for detailed cellular infrmation then you need to use more specific and objective methods like IHC..

I agree with Ahmed that Giemsa stains almost all the cells very well, including mast cells. And yes, you need to befriend a pathologist (like me!).

macrophages, neutrophils, eosinophils, mast cells, lymphocytes can all be easily identified in a good H&E section. Subsets of lymphocytes can't be identified without immunohistochmistry. other noninflammatory cells can also be identified. for the second question Geimsa.

on frozen sections can be easily identified in a good H&E the muscle fibers and their nuclei

D. Meyerholz well summarized the topic.

I can add two suggestions:

1) you can easily distinguish macrophages, even in a PAS positive tissue such as liver (Kupffer cells), carrying out PAS-diastase staining. Macrophages retain purple-magenta stain exclusively;

2) you can find a lot of useful staining protocols on an (also old, I use a 1990 old one) histochemistry book.

Good luck

H&E staining in normal 4-5 µm thick sections : there is a need of knowledge of relevant morphologic features and observer experience. In such case it is possible to recognize basic (immune) cell types, but as was mentioned before - no subtypes of specific cells (for example lymphocytes). Macrophages and foam cells are recognisable in H&E, but much more better in semi-thin sections (2µm) and toluidin blue staining (our experience). It is possible to quantify mentioned cells by means of sterelogic tools. Giemsa-Romanovsky staining is suitable especially for neutrophils, eosinophils and basophils visualisation.

Good luck)

From my experience in tissue samples (not cytology) you can use Giemsa stain for tissue to identifiy leucocyte from thier morphology and to distinguish mast cells. You can use PAS staining for distinguishing plasma cells and I once applied wright stain to tissue sections to distinguish eosinophiles...

A section stained with a well differentiated H&E stain can tell you alot about the pathology of the disease process you are researching. Many different cellular components are distigushable, such as myocytes, fibrocytes, endothelial cells, as well as different inflammatory cells. H&E is the routine stain employed by all pathologists before they request any high complex testing. So, become familiar with a naive artery/ateriole first, so that you understand the structure and function of the resident cells. Once the inflammatory reponse begins in plaque formation, the structure changes and the influx of the types leukocytes varies depending on the chronicity of the lesion. So to answer your question, in a well differentiated H&E section you can determine the presence of lymphocytes, but not the different subtypes, macrophages, infiltrating monocytes, neutrophils,and plasma cells. This is done by the assessing the nuclear morphology and the amount of cytoplasm in the cells. For eosinophils and basophils, I would use a Giemsa stain, just because if the eosin/phloxine is too intense, neutrophils can look like eosinophils, and if the hemetoxylin is too light the basophils can look like a neutrophils. Mast cells can be identified due to the large basophillic granules, but I would recommend confirmation with Toluene Blue staining. Since you are still learning invest in some histology reference materials. Netter's Essential Histology is a good handbook w/ a lot of images to help you classify cell types. And it contains a good diagram of hematopoeisis...important for end stage differentiation of the various leukocytes. For various stains used there are two handbooks used by histologists, Theory and Practice of Histotechnology by Sheehan/Hrapchak and Histotechnology by Carson. There are others, but these three will broaden your understanding of histopathology.

@Elizabeth: well done, thank you for interesting and detailed considerations.

The only spelling error I found in your text (not to teach you!) I would like to correct (hopefuly I am right...)

[ Toluene Blue = TOLUIDINE Blue ] ....right?

Best regards, Wolfgang

I also agree withElizabith, but using quick stains is simple and give excellent morphlogy of most of the cells you are looking for. Such stains include diff quick, rapidiff and RAL.

One can make his own quick stain to cut down expenses.

I do agree Robin Moll that those are macrophages. They can be confirmed by immunophenotyping using CD 68. However certainly one should have the freedom to recognize a proportion of cells without resorting to Immunohistochemistry. And foam cells do seem to come under that category.

Morphological evaluation in any of the Romanowsky stains or H&E will permit identification of almost all types of epithelial and stromal cells.You require some training and experience for the same.Pathologists often diagnose disease by studying morphological alterations on cells.Better you try to get familiar with morphology of these cells.

@Meghanand Nayak

I have never heard of Toluene blue nor does 'Google' pull up any information on it.

It is easy to see how it can be easily misspelled and mixed up.

(Since 1 reply meanwhile has been deleted (Meghanand Nayak) from this thread I have deleted here 2 starting sentences I wrote originally 14th of April 2014) .

@ Cyrus: I thank you really for clarification...{< I guess.....> deleted by commenter)

best regards,

Wolfgang

@ Robin Moll

H&E, Giemsa as well as some of the other stains mentioned will allow one to differentiate between many cell types, provided the observer has sufficient experience. With regards to foam cells, they are quite characteristic morphologically and one should be able to identify them just based on that.

In my previous studies on infection of endothelial cells with cytomegalovirus (CMV) foam cells were often seen in areas where the endothelial cell had sloughed off, perhaps as a consequence of becoming cytomegalic or senesance.

If you are comfortable with your assessment that these indeed are foam cells, you should stick with it but just o prove your point you may wish to do some IHC staining on thse sections.

Chloracetate esterase for neutrophils

Toluidine blue for mast cells.

The majority of cells can be identified readily by routine H&E stain. Giemsa stain used for identifying different types of blood cells. But in doubtful cells you can use other special stains including IHC. In general an open and experienced eyes is always needed for excellent evaluation.

Hi Robin,

Your supervisor sure wants to be on a solid evidence. At PhD level you want to make sure everything is well defended. The responses are very good above, however, cellular morphology can be deceptive at times. Clear cell carciinomas, well differentiated liposarcoma and some other benign tumour cells can resemble those large foam cells under H&E stain.

Of course the site of lesion and age is important. But this is 21st century, so put all your skills and facilities available to prove your findings.

I'd say 99% of all human cells and tissue structures, including pathologic changes can be reliably identified on GOOD quality processed formalin fixed paraffin embedded tissues WITHOUT IHC (immunohostochemistry). The entire science of human pathology is based on H&E. There are specific areas of pathology when the IHC is necessary as an ADDITION to a good quality H&E, i.e. classification of poorly differentiated malignancies, esp sarcomas and lymphomas and some others, like germ cell tumors etc (this is not all inclusive list of all possible rare situations). There still constitute much less than 1% of all human cancers. So, I claim that in 99% of diagnostic cases you don't need any adjuncts like IHC or molecular testing.

Saying that, I have to acknowledge the field of cancer predictive biomarkers, which are completely relying on IHC and molecular studies. Also, the filed of cytopathology is heavily relying on IHC, as many cells (when looked at out of tissue context) are difficult to classify (especially malignant tumors). In contrast, normal cells are usually not very difficult to classify without any IHC for a trained histo/cytopathologist .

Therefore, your question can be answered as above, but it depends on the context and your field of study.

Hi Robin,

PAS stains leucocytes/polymorphs.

Thank you All so much for taking the time to answer my question. I never thought for a moment it would receive such a great response. You have all thrown up some interesting answers and have helped me a lot.

General response

(1) Almost any cell can be identified in a good H&E section if you have the experience or a keen eye and patience.

(2) Befriend a pathologist.

Yours gratefully,

Robin

Good summary.

I agree

Direct first scarlet staining (modified method for Congo red) detects Eosinophils.

Chloracetate-Esterase detects neutrophils, a Giemsa stain also, but all that stuff can be seen in a GOOD differentiated H&E. Key word: good! Every pathologist can tell a bunch of stories about pitfalls due to bad H&E staining,

With H&E stain in a frozen section of 8-10 micron you can identify muscle cell and their nuclei

Using combination of orange red and eosin and hematoxylin will be excellent to identify all eosinophilic cells in paraffin sections. Besides plasma cells, macrophages, clear cells (Helle Zellen), lymphocytes in general, myoid cells, all kinds of muscle cells, neutrophils etc. can be easily identified from their histomorphology easily. Of course a skill in study of histomorphology is preferd.

Pap stain is a very good differential staining method for almost all type of cells.As it contain a mixture of cytoplasmic stain in addition to hematoxylene,the maturation level also can be appreciated in this technique.Also this technique is a regressive staining method so that the crisp chromatin granulation can be helpful for the identification of the cells.However this technique is not advised to dry preparations and samples which contain large amount of background materials.

A Giemsa or Toluidine Blue could solve all the problems related to inflammatory cells.

In addition to the aorta could do with oreceina staining for elastic fibers + masson trichrome staining.

Can anyone following this question point me toward published research that has studied the accuracy of H&E staining (or other routine histology) in comparison to immunological staining? Particularly interested in identification of inflammatory cells.

Thanks

Thank you for all excellent answers. 

could anybody tell me relationship about HE and thermal affects of device on morphological changes of cells? is HE staining adequate to evaluate of morphological changes? or is vital staining more better than HE this issue?

To study morphological changes of cells H&E or any of the stains can be used.

Tolga Karacan - in general the H&E is far superior than a vital stain.

It allows examination of cellular staining, colour, nuclear and cytoplasmic patterns.

Above all it is the most commonly used stain and one can get a permanent record of the slide.

Please collaborate with a histopathologist who is trained (for years) to look at H&E, this will help you to value H&E and appreciate its superiority over other stains, in this respect.

With acid phosphatase you can identify macrophage

With Alkaline phosphatase you can identify neutrophil

With Gimesa stain see attached file and link below

Gimesa stain can done as the followings: Preparation of Gimmsa stain: Stock solution: (4 gm. gimmsa + 250 cm glycerine + 250 cm methanol). Using solution: Take 4cm from stock solution + 96 D.W for staining.

Staining of tissue by gimmsa: Put slide xylene I.II. Then in alcohol 100%. 90%.80%,70% for 3 min in each concentration . Then, wash the slides by D.W. Put slides in the prepared solution (4 cm gimmsa+ 96 cm D.W) for 24 hrs (overnight). Wash slide with D.W very rapid (few second). Rinse the slide in 0.5 % Glacial acetic acid, the color of slide will be turning to red color (rapidly). Wash slide with running tap water. Leaves it dry. Dehydrate in 95% and 100% alcohol rapidly. Put in xylol I.II for 5 minutes for each one and cover with D.P.X

http://www.ihcworld.com/_protocols/special_stains/giemsa_ellis.htm

lymphocytes, macrophages,plasma cells, cant type b and t lymphocytes, eosinophils, neutrophils, marrow myeloid precursor cells,monocytes, mast cells ,megakaryocytes, all look different size, nuclei, cytoplasm and cytoplasmic staining. Epithelial cells from different organs usually look different, germ cells, hepatocytes , mesothelial cells, bronchial epithelial cells, endothelial cells etc etc all look different on H and E. A Giemsa stain will differentiate all cells in a blood film as well.

You can also note the Myoid cells of thymus, Parietal cells (HCl secreting cells) of stomach, blood cells (as already stated), adipocytes, mucous secreting cells in HE section. However there may be some confusion on the identification of mast cells, endocrine cells, cells of transitional epithelium, hepatocytes, as regards there morphology is concerned; of course if you consider their location simultaneously, then it will help the situation....