Week 2 (30/6 - 4/7)

Histopathology

Hi all. For the past 2 weeks, I was attached to the Histopathology lab. Basically the lab does routine work everyday: tissue processing, embedding, microtomy, etc. Of all, trimming can be considered the most interesting process, we will be able to see different tissues such as placenta and tonsils being cut. For every process, it is very crucial that the initial of the staff performing the job is indicated to facilitate traceability, as any small error may result in the wrong diagnosis.

Ok now I shall let you have an idea of how a Histopathology lab works. Following are the procedures that I observe everyday:

Receiving of Specimens – When specimens are first obtained at the lab, the information on the container/plastic bag (patient name/nature of specimen) must be checked against the requisition form. Next, the time and date is recorded and the medtech who performed the check will sign a form for the staff who sent in the specimens to prove the receipt. Labels which contain a barcode unique for every patient will then be printed and stick to the form and the specimen.

Most specimens sent in are fixed in formalin, which hardens the tissue for easy trimming. Large specimens are usually fixed overnight before trimming to ensure that formalin penetrates into all parts of the specimen. In the lab, formalin prepared would be sufficient for one week use.

Trimming and Passing – Before trimming of some large specimens such as breasts, asst pathologists will dye the specimen with different colored dyes to indicate the margins. While trimming, they will dictate the measurements and observations seen on the specimen (eg. a tumor measuring 2cm by 2cm is seen at 7 o’clock position) and these are immediately typed out by a lab assistant who sits just beside the asst pathologist. These observations would be printed out, attached with the requisition form and sent to pathologists to make the diagnosis. They will cut certain parts of the specimen where they suspect an abnormality may be present and ‘pass’ them into cassettes labeled with the patient number and year. For small samples, the number of strips of tissue will be written on the cassette to ensure that the embedder removes every single piece of tissue. Gel is also used to clump all the small strips of tissues together. Cassettes are labeled using a special marker which is resistant to formalin and other reagents. Also, different colored cassettes are used for different cases. All the cassettes are then placed into the automated tissue processor which works overnight, to perform thorough fixation as well as dehydration to remove all water from the tissue.

Embedding – First job performed every morning, after cassettes are removed from the processor. Tissues are first transferred to an embedding mould according to the size and pressed down using a weight (to ensure that the tissue will be flat), and embedded with paraffin wax. Blocks are then put onto an ice block for cooling.

Shaving, Sectioning and Fishing – After the wax is hardened, blocks are shaved at 10µm using the microtome to first expose the tissue. This is followed by sectioning, where tissues are sectioned at 3µm. Sectioned tissues are first placed into cold water and fished using pencil-labeled slides before transferring into the water bath containing hot water (43-48 deg C), which expands the tissue to remove folds. A thermometer must be placed inside the water to ensure that the water is not too hot as this may damage the tissue. The orientation of the sections is also very important in fishing. If fished too high, the tissue may not be stained by the autostainer (reagents in the autostainer are only of certain volume). Slides will then be arranged in slide holders and placed into the oven for heating for 15 mins at 85 deg C to ensure tissues are properly attached onto the slides to prevent detachment/sliding off during staining. Tissues can be fished 6, 3, 2 or 1 in a slide according to the size (smaller tissues are fished more in a slide).

Staining – Slide holders removed from the oven is then immediately placed into the auto-stainer, which performs the H & E stain. Special stains such as PAS, iron and reticulin stain are performed manually. Different programs in the autostainer perform different procedures (eg. program 8 is used for H & E staining and program 7 is used for dehydration, for special stains). This machine is connected to an autocoverslipper, which automatically mounts coverslips on slides without producing bubbles. Many racks can be loaded into the autostainer at once, thus the use of this machine had saved a lot of time compared to manual staining.

Sorting – Slides stained will then be checked against the tissue blocks (to tally the size and shape of tissue) to ensure labels are pasted correctly before they are sent out to the pathologists, who make the diagnosis. Used blocks will be kept into boxes in ascending order to ensure easy retrieval when additional special stains or re-cuts are requested by pathologists.


Other Histopatological Aspects

Frozen Sections – This is done when immediate results are required, usually when an operation is still in progress. When the doctor removes any tissue and is unsure whether the surrounding tissues are affected, they will request for frozen sections. Once the histolab receives a frozen section request from the operating theater (OT), a medtech would have to rush to the specimen room located around the OT to collect the fresh specimen. The pathologist in charge should already be on stand-by in the lab. Once the specimen reaches the lab, the pathologist will carry out the trimming personally. The trimmed tissue will then be treated with liquid nitrogen and sectioned using the cryocut. This is followed by the manually performed rapid H & E staining and microscopic analysis by the same pathologist. They will then call the OT to report the results. The whole process (from the time when the specimen reaches the lab to the delivery of results) must be completed within 20 mins, thus it can be very stressful when performing this procedure.

Immunohistochemistry Stain – Commonly used in the diagnosis of abnormal cells such as those found in cancerous tumors, this type of stain is performed when the pathologist suspects any abnormality in the morphology of the H & E stained slides and requires confirmation. The stain exploits the principle of antigen-antibody reaction; using specific antibodies to detect the antigens present in the cells. Firstly, slides with the cut sections and cover tiles (to protect the tissue from any damage during the staining process) are loaded into the machine. Then tumor markers such as CD31, estrogen and CK-7, which are contained in containers, are added. Positive reactions with these markers can indicate particular cellular events such as proliferation or apoptosis. These positive results can be visualized based on enzymatic conversion of an added substrate (DAB) into a brown colored precipitate at the sites of antigen localization (ie. if cells stained appear brown, there is a positive antibody-antigen reaction).

Special Stains – These are done also as confirmatory tests for diagnosis. When the pathologists suspect an abnormality in the H & E stain, they will order for special stains. Some examples are PAS & PASD, GMS and TB and Prussian blue stain (I will only elaborate on PAS & PASD, cos this is the most commonly used test):

PAS (Periodic Acid Schiff’s) detects the presence of glycogen, fungus and mucin. It stains these components magenta (using Schiff’s reagent) and the nucleus blue (haematoxylin as counterstain). PAS is always done with PASD, which has the same protocol as PAS; only that it has an additional step of treatment with diatase, which deploys glycogen into smaller sugar units that can be washed out after treatment. Glycogen stained magenta on the PAS stained slide and will be absent on the PASD stained slide. This absence of glycogen will clearly show the presence of fungus and mucin.


I will be changing lab next week. But I havent seen post mortem case (autopsy of babies)! Cos there is none. Not that I'm a saddist, my supervisor say must see. Nvm, this might not be a bad thing.

Anyway that's all I have for now. Have fun =)


Ka Hang
TG02