1. Describe and explain the need for different tissue preparation techniques for the following samples:
a) Native renal biopsy
1mm biopsy for EM should be taken from each end of the core biopsy if it is larger than 7mm. EM is taken from each end as we are unsure what end contains glomeruli as it is unoriented. The EM sample is placed in 2.5% buffered glutaraldehyde then changed to osmium tetroxide after 24 hours. EM is performed to evaluate the morphology of the glomerululi; the basement membrane, any immune complex deposits or glomerular changes which cannot be assessed with a H&E stain. The remainder is placed in formalin for paraffin embedding. The biopsy should be fixed for at least 1 hour before processing.
b) Skin biopsy for blistering skin disease
Two biopsies are taken; one skin sample is placed in formalin for processing into a paraffin block and cut as any other routine sample. The other skin biopsy is placed in Michel’s solution (a transport media) for immunoflouresence (IMF). Contact with formalin for the IMF sample should be avoided at all times as it can produce autoflouresence (causing false positives). The IMF sample is frozen and cut on a cryostat and stained to demonstrate specific immunoglobulins to aid in the diagnosis of blistering diseases such as Pemphigus and Pemphigoid.
c) Bone marrow trephine
Bone marrow trephines are fixed in formalin and are placed into EDTA decalcification fluid for 48 hours in a 37oC oven. EDTA is a chelating agent; it removes calcium from the sample making it easier to cut on a microtome. EDTA is used as it protects the morphology of the cells as well as allowing immunohistochemistry to be reliably preformed later on the tissues once they are then processed into a paraffin block.
d) Whole, fresh lymph node
These are sent to the lab fresh, normally urgent as results from the initial investigation can determine the immediate action of the surgeon if the patient is still on the operating table. They are described by a pathologist who then selects suspicious or representative parts to be cut fresh on a cryostat or to take imprints to be stained. Microbiology could be taken to query infection or flow cytometry for immunophenotyping to locate any tumour cells. Once the initial investigations are complete the tissue is then fixed in 10% formalin, sampled and processed. The sections are cut at 2µm this is because the lymph node is highly cellular so the sections are cut thinner to attempt to section 1 cell thick.
2. Give examples of types of samples that must be dealt with urgently and why.
Potential renal donor biopsies, these are urgently processed the same day and given to the pathologist to assess whether the kidney is healthy and can be transplanted. This is urgent as the kidney will start to deteriorate if left too long. This is one of the services performed out of hours by the on-call service.
Query Hirschprungs disease biopsies from babies born recently with clinical details of not passing meconium. These are processed for the next day urgently with a Hirschprungs serial, which is 20 slides with 6 sections on each. They are processed urgently as the baby may require a stoma to be placed. For a diagnosis to be obtained appropriate biopsies should be sent from the affected area of the rectum a special stain should be performed on the frozen tissue; an Acetylcholinesterase to confirm the diagnosis.
Frozen sections must be dealt with urgently as the patient is on the operating table. For example during kidney transplant if a nodule is found on the soon to be donar kidney then it needs to be investigated before transplant. The affected tissue is removed and before the transplant can be carried out the surgeon must guarantee that the tissue is not diseased, this is done by a frozen section. The surgeon takes a small section of the tissue for diagnosis, the pathologist describes the biopsies are orientates them for the biomedical scientist (BMS). The BMS then snap
