Evaluation

Author: Mikael Häggström

Microscopy settings

File:Light microscopy with and without condenser.jpg

Light microscopy with and without condenser. At low magnification, using a condenser may limit the field of view, and in such cases it is preferable to not use it. At high magnification, a condenser makes borders less marked, and is generally preferable in such cases.

File:Calcium pyrophosphate dihydrate crystals without and with condenser, annotated.jpg

An example of a situation where microscopy without condenser is preferable at high magnification is the evaluation of crystals (calcium pyrophosphate dihydrate crystal deposition disease pictured).

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. 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.

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):

1. Use high magnification and focus on a specimen using the main focus knob.
2. 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.

Main steps

• Preferably, look up past medical history of the patient, mainly past cancers that could possibly appear in the current specimen.
• Look at each microscopy slide by eye, to plan the microscopy screening so as to not miss peripheral fragments.
• Have a systematic direction of screening 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.

• 
  Example starting position of objective.
• 
  Example slide scanning directions.

While learning, you will generally focus relatively more on high magnification features with high specificity, but still have a habit of learning how your cases look at low magnification as well. 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.

Complete your evaluation even if you encounter a finding, as there may be additional findings as well.

Artifacts

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:

• 
  Cellulose contamination, here seen on H&E stain and polarized light, respectively. In contrast to foreign bodies that were truly in the specimen, contamination overlaps with or disrupts tissue more unnaturally.
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  Cardiac muscle (bottom) with contamination from thyroid tissue (center).
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  Crush artifact from compression by forceps on the tissue sample.
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  Folding artifacts (white arrows) and a crush artifact (black arrow, with cytoplasmic hypereosinophilia and nuclear pleomorphism) from a needle.
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  Tearing artifacts, such as can be caused by:
  - Microtomy with a nick or blemish in the knife edge.
  - Traction of the sections.
  - Too much or too little alcohol dehydration.
  - Sectioning calcified parts, which can be decalcified or removed.
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  More tearing artifacts, showing that they may be more circular than fusiform.
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  'Formalin pigment artifacts
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  Air bubble entrapment artifacts
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  Staining artifacts by residual wax, resulting in pale areas where cellular structures are not discernible.
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  A separation artifact in top image makes the tumor look incompletely excised, but the next microtomy level (bottom image) shows a surgical margin of connective tissue.

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.

Inflammation

• Neutrophils generally confers a diagnosis of acute inflammation.
  Neutrophils generally confers a diagnosis of acute inflammation.
• Plasma cells and lymphocytes generally confers a diagnosis of chronic inflammation if present in increased amount for the location.
  Plasma cells and lymphocytes generally confers a diagnosis of chronic inflammation if present in increased amount for the location.
• However, disregard intravascular white blood cells.
  However, disregard intravascular white blood cells.

Measuring distances

• You can use a regular transparent ruler for most purposes, since it is rarely necessary to specify distances as more exact than millimeters.
  You can use a regular transparent ruler for most purposes, since it is rarely necessary to specify distances as more exact than millimeters.
• Alternatively, use a marking pen to mark the limits under the microscopy, and then use a regular ruler to measure the dimensions. This works even if the distance exceeds the field of view, or spans more than one slide.
  Alternatively, use a marking pen to mark the limits under the microscopy, and then use a regular ruler to measure the dimensions. This works even if the distance exceeds the field of view, or spans more than one slide.
• If sub-millimeter measurement is needed, you may use a calibration slide, which is basically a translucent ruler with 0.1 mm line resolution or smaller, which may cost about $15 if none of your colleagues has one to lend, or the department can't provide one for you.
  If sub-millimeter measurement is needed, you may use a calibration slide, which is basically a translucent ruler with 0.1 mm line resolution or smaller, which may cost about $15 if none of your colleagues has one to lend, or the department can't provide one for you.
• To measure distance with a calibration slide, put it on top of the tissue slide and switch focus between the two.
  To measure distance with a calibration slide, put it on top of the tissue slide and switch focus between the two.

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.

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 mm)² x 0.79

• Micrograph of a calibration slide, showing the diameter of the field of view. On this slide, 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 (and thus an area of 0.24 mm2).
  Micrograph of a calibration slide, showing the diameter of the field of view. On this slide, 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 (and thus an area of 0.24 mm²).

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
Olympus BX50, BX40 or BH2 or AO or Nikon with 15x eyepiece Olympus BX43 with 10x eyepiece	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
	0.096

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
	0.096

General notes

Further reading: Starting pathology (handbook) Learning pathology Emergency pathology Fixation Gross processing Evaluation: Suspected malignancies Immunohistochemistry Consultation Reporting Secrets

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