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Pathology & Immunology

Houston, Texas

Pathology and Immunology
Pathology & Immunology
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Submitting specimens for EM study

Proper specimen fixation for EM is a two step process. First the tissue is fixed for an hour in 3% gluteraldehyde. It is then rinsed in buffer and fixed for an additional hour in osmium Tetroxide. It is then rinsed, dehydrated through a graded series of ethanol, and embedded. The first step is best performed by the person(s) collecting the specimen. We can provide you with vials of the gluteraldehyde ready to use. You collect the tissue sample to be examined in the vial(s), label it properly (See below.), and send it to the Baylor Pathology E. M. Lab. We will complete the fixation in osmium and perform the embedding and sectioning steps.

  1. The tissue taken must be representative of the disease process. For example, in order to diagnose nephrotic syndrome, renal cortex containing glomeruli must be submitted. The necrotic centers of tumors usually do not yield useful information. Choose an area in which the cells are most viable.
  2. In order to allow rapid fixation, at least one dimension of the tissue to be fixed must not be greater that 1 millimeter. It is better to dice the tissue into cubes not greater that 1 millimeter on a side. It is also important not to crush the tissue when handling it. Use the sharpest blade available; do not squeeze the tissue with forceps.
  3. The tissue must be put into chilled E.M. fixative as soon as possible after being removed from its blood supply. The initial hour of fixation should be done on ice, but DO NOT EVER ALLOW THE TISSUE TO FREEZE. If there is any thought about doing E.M. on the case, fix tissue for E.M. right away. Tissue fixed in formalin, or even embedded in paraffin, subsequently can be prepared for E.M., but the results will not be nearly as satisfactory, and may not be useful at all. Tissue that has been sitting in a bucket or on the bench for any length of time (depending of the tissue) will almost assuredly be useless. The E.M. Lab will furnish vials of fixative on request. Do not send unfixed tissue of any kind to the E.M. Lab.
  4. Submit the specimens to the E.M. Lab as soon as possible. Glass scintillation vials with about 5 mls. of fixative are ideal, but any vial that will be leak proof in shipping will do. You can "hold in Fix" until you are certain you want E.M. done; however, the tissue will deteriorate over time. It is better for us to "Block and Hold" the tissue because then the fixation will be complete and optimal. Fixation for E.M. is a two step process. Glutaraldehyde fixes proteins, but lipids, a major component of membranes and other cell structures, are not fixed until the tissue is put into osmium.
  5. Label the specimen vial carefully with the patient's name and the specimen's surgical number and fill out the middle portion of the E.M. diagnostic request form completely. (See sample form.) The E.M. Lab will not process the specimen unless this form is filled out completely. These forms are available on request from our lab.

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