On this page, we give an overview of the tissue/slide preparation process so participants that do not have prior experience in analysis of histopathology images have an overview. We also give a short introduction to mitosis counting, i.e., how it is performed in standard clinical practice and what the challenges are.
After breast tumor excision is performed in the operating room, the excised material is sent for analysis in a pathology lab. The tissue preparation process starts with making smaller cuts of the material that are then fixed in formalin and (after processing) embedded in paraffin.
Using a high precision cutting instrument (microtome), thin sections are cut from the paraffin block, which are then put on glass slides. The final stage of the tissue preparation process is the staining of the sections with stains that highlight specific structures of the tissue so they are better visible under a microscope.
The standard staining protocol uses the hematoxylin and eosin stains (the diagnostic/prognostic procedure for all patients always starts by staining the sections with these stains). The hematoxylin dyes the nuclei a dark purple color and the eosin dyes other structures (cytoplasm, stroma, etc.) a pink color.
From top left to bottom right: 1) Small cuts are made from the tissue 2-3) The smaller cuts are put into cassettes and (after processing) embedded in paraffin 4) Thin sections are made from the paraffin blocks with a microtome 5) The sections are put onto glass slides for staining 6) H&E stained slide
Recent years have brought the trend of digitization of histological slides. Digital slide scanners, in combination with digital slide viewers, aim to provide the experience of viewing a digital slide on a computer monitor in a manner analogous to viewing it under a microscope, but with all the added benefits of the digital format (ease of annotation, image analysis, collaborative viewing etc.).
The output of the digital slide scanners are multi-layered images, stored in a format that enables fast zooming and panning. Depending on the area of the tissue that is present on the slide and the magnification and resolution at which the slide is scanned, the lowest layer of the digital slide can be up to several tens of thousands of pixels in width or height.
Currently, digital slides are mainly used for research, education and remote consultation purposes. Their use for routine diagnosis and prognosis is not yet common, however that is expected to change in the coming years. Availability of automatic image analysis algorithms that can aid pathologists in their work can be a major incentive for acceptance of digital slides in the routine pathology lab workflow.
The Aperio ScanScope XT scanner model used at our Pathology Department
Mitosis counting and scoring
Histological tumor grading systems assess the differentiation of the tumors, i.e., how closely they resemble normal tissue when examined under a microscope. Generally, patients with well-differentiated tumors have better outcomes and vice versa. Clinicians use the histological grade, among other factors, to give an estimate of the patient’s prognosis and develop individual treatment plans. The most widely used system for histological grading of invasive breast cancer (BC) is the Bloom & Richardson grading system (B&R). It consists of assessment of three components: nuclear pleomorphism, degree of tubule formation and mitotic activity.
Mitotic activity is one of the strongest prognosticators for invasive breast carcinoma. It is expressed as the number of mitotic figures per tissue area. Aggressive tumors have a high proliferation rate, which is reflected in a high number of mitotic figures present in the histological sections. As part of the B&R grading system, mitotic activity is routinely assessed in pathology labs across the world. In addition, the mitotic activity can be used as a prognosticator independently of the B&R grading system.
Typically, the pathologist recieves a panel of slides for each case that is to be graded. He or she then proceeds to select one slide where the histological grading will be performed. The mitosis counting is performed in 8-10 consecutive microscope high power fields (depending on the microscope model) that should correspond to an area of 2 mm2. The standard guidelines are to select an area that encompasses the most invasive part of the tumor, at the periphery and with highest cellularity. Depending on the number of figures counted, a mitotic activity score is assigned. Cases with 6 or fewer mitotic figures present are assigned score 1 (best prognosis). Cases with more than 10 mitotic figures are assigned score 3 (worst prognosis). The intermediate cases are assigned score 2.
Challenges in spotting mitotic figures
Because of the aberrant chromosomal makeup of many tumors (aneusomy, polysomy, translocations, amplifications, deletions), the appearance of mitotic figures in the images can significantly differ from the textbook examples of a splitting nucleus. In addition, imperfections of the tissue preparation process result in tissue appearance variability, which can present a challenge for an automated mitosis detection system. In the image below, some examples of mitotic figures in H&E breast cancer sections are shown.
Examples of mitotic figures (marked with green arrows)
Most commonly, mitotic figures are exhibited as hyperchromatic objects. In addition, they have absence of a clear nuclear membrane, “hairy” protrusions around the edges and basophilia instead of eosinophilia of the surrounding cytoplasm. However, these are more guidelines than hard rules, and the bulk of the training of pathologists is done by looking as specific examples of mitotic figures.
One of the main challenges in spotting mitotic figures is that other objects such as apoptotic nuclei (shown on the images below) can have similar appearance, making it difficult even for trained experts to make a distinction. Lymphocytes, compressed nuclei, “junk” particles and other artifact form the tissue preparation process, can also have hyperchromatic appearance.
Examples of apoptotic nuclei, most commonly mistaken as mitoses (marked with green arrows)