Pathology
Disclaimer: All of these primers are written by parents that have NO medical background prior to having to deal directly with the disease. The contents should not be misinterpreted as medical advice. Our opinions could be wrong. The intent is to help you come up the learning curve and hopefully bypass some of the difficulties we have encountered. Always refer to your doctor. Like almost all things in life, there are many correct answers.Note: Attempted to highlight 'Why You Care' in purple.
For most of the Histiocytic Disorders you end up getting a tissue biopsy - a small sample is taken and examined to help with diagnosis. It really helps if you have a basic understanding of how this whole process works. I wish I would have had this fundamental understanding when we started all this. It is also critical to understand pathology and the pathology of histiocytosis in particular because laboratory and clinical findings are insufficient for diagnosis. It is necessary to biopsy involved tissue to make the diagnosis in almost all cases. If you are just learning that you or someone close to you may have histiocytosis, you are probably going through what is called differential diagnosis - trying to determine which of several diseases or conditions with similar symptoms are involved. Pathology & Histology attempt to help answer these questions (along with clinical presentation, staging and/or symptoms).
Definitions You Need First Methods of Pathology Viewing Methodologies Staining Methodologies Tissue Preparation Methodologies Steps to Get Ready for Microscopy Interpreting a Pathology Report Comparison Between LCH, JXG & Others
Definitions
Biopsy - Removal of a small amount of tissue for examination. Note that there are several methods of removal and there are several methods of handling, treating and examining the tissue after removal.
Differential Diagnosis - The determination of which two or more diseases with similar symptoms is the one from which a patient is suffering from based on an analysis of the clinical and pathology data.
Pathology - The study of disease. In this context it is the study of the diseased or dysfunctional tissue.
Histology - Branch of anatomy that studies tissue, typically used as a synonym for microscopic anatomy.
Cytology - The study of the cell (usually at the microscopic level).
Pathology, Histology and Cytology can almost be used as synonyms in our context.
Ways to View Tissue & Cells
There are several different ways to look at tissue, we will look only at three main methods. A basic knowledge of these methods will help you interpret pathology reports and help you understand why different types of biopsies might need to be done.
Light Microscope - The first, most basic and most common type of microscopy. Able to see inside cells when staining is used. You can look at a tissue sample at several different magnifications. At a lower power you would see a large cellular field that could contain hundreds of cells. Image 1 below is an example of Langerhans Cell Histiocytosis showing many cells. It can be considered a gross view of the lesion. Images are typically in color and appear as cross sections of cut tissue.
Transmission Electron Microscope - Produces the most detailed images. Image 2 below is an example of a common macrophage. In image 3 below you can see an extremely high resolution picture of one feature known only to be found in a Langerhans Cell, the Birbeck granule. EM is more costly, requires 'special' preparation and sometimes requires a greater amount of tissue. Images are typically black and white and show more detail than a light microscope.
Scanning Electron Microscope - Produces surfaces images of cells. Image 4 below is a quite popular image of a macrophage (gray) about to eat or phagocytise a bacteria (yellow). The colors are artificially added. Images typically look 'three-dimensional' and can be in black and white or artificially colored.
Others - There are many other methods that are variations on these main themes. They are not discussed here because most of them are not used for diagnosis, they are instead used for research. Here are some examples: phase contrast microscopy, polarizing microscopy, confocal microscopy, fluorescence microscopy, Radioautography, living tissue examination.1) 2)
3)4)
Staining Methodologies
Tissue tends to be clear in thin sections. There are several ways to make the tissue visible under a microscope. Understanding these methods will help you understand what types of stains need to be done and how to interpret your pathology report.
Basic Staining - Basic stains are the oldest and most common method of staining. The stains are attracted to different acidic and basic components of cell. Other basic stains differentiate between the fibrous and non-fibrous parts of a cell. Some other stains precipitate on tissue and form metallic deposits. The red blue and purple images (see image 1 above) are typical of basic stains.
Histochemistry & Cytochemistry Stains - These stains react with large molecules (macromolecules) present on, in and around the cells.
Immunohistiochemistry and Immunocytochemistry Stains - Uses the specificity of the immune system. Antibodies are paired with colored indicators that match with the antigens presenting on the cells surface. These stains can be incredibly specific reacting only on cells that are presenting a specific antigen. This property gives pathologists the ability to tell cells that look alike but function differently apart.
At first it might not seem necessary to learn about how tissue is prepared, but if you do not know how the biopsy tissue is going to be used, you may not end up getting the correct amount or kind of tissue sample. Or if the tissue is not prepared correctly, you may not be able to examine it the way you need to (for electron microscope for example). A basic understanding might empower you with the ability to ask the right questions at the right times and this may allow you to cut critical time off the diagnosis and possibly prevent repeat biopsies.
Fresh Tissue - Sometimes a 'fresh tissue' sample is ordered. This is done if the doctors, surgeons or pathologists are not sure how to treat the tissue. Tissue is placed in a preservative. It can be kept in this state around 30 days.
Paraffin Block - This is the original and most common method of embedding tissue.
Plastic Resin - Instead of paraffin, a resin is used. The benefit is that the tissue can be placed in the plastic at room temperature. This is not true for paraffin, the tissue has to be placed into hot melted paraffin. Resin avoids the shrinkage caused by the high temperatures for paraffin.
Steps to Get Ready for Microscopy
These are the general steps and they differ (chemicals, machines used) in detail at each step depending on the type and end usage goal (EM, light).Biopsy - Acquire the tissue Fixation - Preserve tissue from degradation Dehydration - Remove water Clearing - Infiltrate with solvent Embedding - Place in plastic or paraffin Sectioning - Slice into thin sections (sometimes the block is frozen first to prevent the loss of lipids and to make it more rigid for cutting) Mounting - Place on a clear base for easy handling Staining - Add stains to make the clear tissue show color
Interpreting a Pathology Report
There are several recurring themes you may see in a pathology report. I have learned from experience that you need to read a pathology report several times and dissect what they are really saying.
Large Sections - This is an overall review on how the tissue looks as a whole. See image 1 above for an example.
Morphology - Refers to how a particular cell looks.
Staining - When stains are applied (see staining methodologies). The pathology report will contain references to whether a stain was positive or negative.
By taking the picture as a whole, i.e. what does the lesion look like in section, what do individual cells look like and what immunohistiochemical markers are positive and negative, couple all of this with the clinical presentation and it is usually possible to diagnose a disease or at the very least say that it is not a certain disease. You also have to remember that the results are only as good as the tissue sample. Sometimes a patient may have a disease, but the lesion or tissue site is not 'active' and is therefor hard to diagnose.
Pathology Comparison - JXG Vs. LCH
More to come here. There is still a ton to learn on how to exactly understand distinguish the different dendritic cells and macrophages involved in Histiocytosis. Immunohistiochemistry research is doing a lot to advance this area. For now, here is a pathology comparison table and a list of key pathology quotes from technical sources.
