- 1 Microscopy settings
- 2 Priority
- 3 Main steps
- 4 General patterns
- 5 Inflammation
- 6 Marking slides
- 7 Measuring distances
- 8 Counts per mm2
- 9 Artifacts
- 10 Micrography and telepathology
- 11 Cytology
- 12 Consideration
- 13 Notes
- 14 Main page
- 15 References
- 16 Image sources
Generally the condenser is placed in its highest position or just slightly lower. At low magnification objectives (mainly 4x and 10x objectives), the opening of the condenser (or iris) diaphragm should be wide open. This corresponds to turning away or "lowering" the condenser on microscopes where the condenser apparatus can be turned to the side (and is shown as "without condenser" in images below). For high-dry (40x) and oil-immersion objectives (100x), the diaphragm should be closed slowly while looking at a sharply focused section until the level of illumination is just slightly reduced, in order to attain optimal contrast and resolution (and corresponds to "with condenser" in images below).
Low magnification has a greater span of focus compared to high magnification, so it is normal to need to focus if you're increasing magnification. However, if you find that you need to change focus even if going from high to low magnification, try the following (if you can adjust the eye piece):
- Use high magnification and focus on a specimen using the main focus knob.
- Switch to low magnification, and focus using the eye piece adjustment.
If there's a constant visual artifact, even after you've cleaned the eye piece and objective lenses with lens tissue, try raising or lowering the condenser if you can, and the artifact may disappear out of focus.
Whenever you have more than one case, generally have an initial look at the requisition form and/or in the microscope at the most likely relevant area of each case, and prioritize the case(s) that likely need to be done first. Indications to prioritize case(s) are mainly:
- Clinical management of the patient highly benefits from a fast diagnosis.
- Being marked as a rush case.
- There's a significant probability that it will require further workup such as immunohistochemistry, especially when there is an impending deadline for ordering it.
- The optimal person to ask for advice may not be available later. Further information: Consultation
For a pile of many cases of relatively low risk of a need to prioritize any one over another, such as gastrointestinal biopsies, a very quick glance at the forms and/or a naked eye look at the glass slides is generally acceptable.
- Preferably, look up past medical history of the patient, mainly past cancers that could possibly appear in the current specimen.
- Try to pick up glass slides without touching the tissue area, as fingerprints may interfere with the evaluation.
- Look at each microscopy slide by plain eye, to plan the microscopy scanning so as to not miss peripheral fragments.
- Have a systematic direction of scanning through microscopy slides, such as from top left to bottom right as seen in the microscope. When the microscope makes what you see two-way mirrored, the starting position is with the objective pointing at the bottom right of the glass slide. You may center on findings on interest and evaluate them at higher magnification, and then resume the scanning
- Look in particular for whatever is requested or suspected on the requisition form or equivalent. For each type of condition, initially you will generally focus relatively more on high magnification features with high specificity, but you should still have a habit of looking at low magnification as well to get an idea of its pattern. In time, you will increasingly correlate diseases and conditions with their overall low magnification patterns - patterns that may require 1000 words to describe and thus cannot conveniently be part of written criteria, but will nevertheless allow you to make quicker and more accurate diagnoses, or at least clues thereof.
- When you find something suspicious, it is helpful at least in the beginning to evaluate them systematically by a low-to-high magnification approach (example below showing a basal-cell carcinoma):
- Complete the scanning even if you encounter a finding, as there may be additional findings as well. It is usually helpful to Google multiple images of the normal histology of the location you are looking at, and compare those to what you see, and focus on whatever features may deviate from the normal.
Following are major patterns that often help in making a diagnosis.
When feasible, classify nuclei as follows:
Pleomorphic when having different sizes and shapes. This often correlates with an increased nucleus to cytoplasm ratio. These features generally favor malignancy in the evaluation of suspected malignancies.
- Patterns of nuclear disintegration
...Then slowly approach the slide while making sure that the tip remains in the location you want to mark. Be careful not to obscure findings of interest with the mark. If the slide is likely to undergo whole slide imaging, make the mark on the back and not on the coverslip (as it reduces the risk of erroneous focusing of the cameras).
There are also eye pieces that show a ruler in the field of view, but make sure you match it with the correct objective and other settings to make the measurement valid. A calibration slide is more robust because of such sources of error.
Counts per mm2
There are multiple situations where a finding will be quantified in terms of amount per mm2. To make such calculations, you need to know the size of the area you see in the microscope. It is usually possible to look up what theoretically would be the area, but the most reliable way of knowing is to use a calibration slide to measure the diameter of your view. The area is then calculated as:
- Area in mm2 ≈ (diameter in mm2) x 0.79
On the imaged example, each square is 0.05 mm wide, and each line represents 0.01 mm, making the diameter of the field of view 0.55 mm in this case. Thus, you can calculate the area in mm2 by Googling:
- 0.55 x 0.55 x 0.79
- (equals approximately 0.24 mm2).
Generally count at least 10 fields of a high power view (or more if specifically instructed). You can keep the count in your head by repeating the total count and the order of the area you are looking at, such as:
- "Zero (instances) of one (field of view)"
- "One of two (as you see one instance as you've moved to the second field of view)"
- "Two of two" etc.
Subsequently, the count per mm2 is calculated as follows:
|Count per mm2 =||Total count|
|Number of fields x Area per field (in mm2)|
For example, if you reached "15 of 10", and the area of your field is 0.2 mm2, the count per mm2 can be calculated by Googling:
- = 7.5
Sometimes "high power field" (HPF) is used for area, but it has a substantially different area for different microscopes, for example:
|Microscope type||Area per HPF|
||0.096 mm2 |
|AO with 10x eyepiece||0.12 mm2 |
|Nikon Eclipse E400 with 10x eyepiece and 40x objective||0.25 mm2|
|Leitz Ortholux||0.27 mm2 |
|Leitz Diaplan||0.31 mm2 |
When your instructions are to count a specific number of HPFs, one HPF can be assumed to be 0.2 mm2. If the view area in your microscope significantly differs from this area, calculate how many views you need to count as:
|Views = HPFs required x||0.2|
|Your microscope area (in mm2)|
For example, if your instruction is to count 10 HPFs and each view in your microscope shows 0.096 mm2, you should count in this many views:
|10 x||0.2||≈ 21|
Subsequently, if your microscope area is significantly different from 0.2 mm2 and you need to state your result in terms of count/HPF, use:
|Count/HPF = Average count in your view x||0.2|
|Area of your view in mm2|
For example, if you have counted an average of 10 cells (or other object of interest) in each of your views, and the area of your view is 0.096 mm2, then your count/HPF is:
|10 x||0.2||≈ 21|
In microscopy, an artifact is an apparent structural detail that is caused by the processing of the specimen and is thus not a legitimate feature of the specimen. Major artifacts to account for include:
Differential diagnoses of artifacts are mainly:
- Foreign bodies. In contrast to contamination, these conform more naturally to the surrounding tissue.
- Organisms, to be particularly considered when there are multiple objects of the same size.
Order recuts from the same paraffin-embedded tissue if artifacts significantly impairs your diagnostic evaluation of the glass slide. However, artifacts caused by gross processing may affect recuts as well.
Micrography and telepathology
Unless you have more specific equipment for taking microscopic images and showing cases to remote colleagues, you can perform these tasks as follows:
- With a stationary computer, you can connect to a microscopy camera. For telepathology, you can start a videoconferencing session with your senior, then share the screen while showing a micrograph, or the live view so that you can move around.
- With a mobile phone:
- You can send micrographs to your senior, but it is technically difficult to keep the focus while moving the glass slide.
In cytology samples, or any sample with scattered cells rather than coherent tissue, also evaluate the following:
- Adequacy of specimen. Cells may be too few or too obscured by other material to make a proper diagnosis.
- Background, mainly if it is clear or dirty
- Overall cellularity
Further information: Cytology
First, generally suspect the common conditions for the location at hand. A less common variant of a common condition is often still more likely than a rare condition, so generally call the latter only if it really fits the picture.
- Patrice F Spitalnik. Histology Laboratory Manual, Vagelos College of Physicians & Surgeons Columbia University. Retrieved on 2021-09-20.
- . Infiltrating Ductal Carcinoma of the Breast (Carcinoma of No Special Type). Stanford University School of Medicine. Retrieved on 2019-10-02.
- Klimstra, David S.; Modlin, Irvin R.; Coppola, Domenico; Lloyd, Ricardo V.; Suster, Saul (2010). "The Pathologic Classification of Neuroendocrine Tumors ". Pancreas 39 (6): 707–712. doi:10.1097/MPA.0b013e3181ec124e. ISSN 0885-3177.
- Taqi, SyedAhmed; Sami, SyedAbdus; Sami, LateefBegum; Zaki, SyedAhmed (2018). "A review of artifacts in histopathology ". Journal of Oral and Maxillofacial Pathology 22 (2): 279. doi:10.4103/jomfp.JOMFP_125_15. ISSN 0973-029X.