Endomyocardial biopsy is still the gold standard for monitoring the allograft. Biopsies are also frequently performed to determine the etiology of heart failure in nontransplanted patients. The tissue is usually procured with a bioptome through either the jugular or the femoral vein. There is evidence that with three pieces only 95% of inflammatory infiltrates are detected. How-ever, if four pieces are examined, up to 98% of infiltrates are detected. The working formulation for heart allograft monitoring therefore recom-mends examination of at least four pieces of tissue.4Documenting the number of biopsy spec-imens received is therefore important. The speci-mens are handled differently depending on the timing or the reason for the biopsy.
Following a few simple rules ensures optimal preservation of the tissue for diagnostic analysis. Minor modifications to these rules for specific tests or research protocols can be made without disrupting the work flow in the heart biopsy suite. Some helpful hints include the following.
1. Plan ahead. Take into account that the work-ing formulation recommends “four to six undi-vided pieces of tissue,” “one piece frozen,” and “no tissue routinely fixed for electron micros-copy.” Commonly, the fixative of choice is 10% phosphate-buffered formalin. Alternatively, fix-ation can be done in glutaraldehyde for micros-copy or in other fixatives that preserve antigens for immunohistochemistry studies.
2. The tissue should not be handled with forceps or divided with a scalpel. The tip of an intravenous catheter or syringe needle is usu-ally a good instrument for picking up the bi-opsy. Squeezing the tissue can produce artifacts that upon microscopic examination render it un-interpretable.
The tissue should be fixed immediately in the desired fixative that has been allowed to reach room temperature. Cold fixative enhances contraction band artifacts. The tissue should not be allowed to sit for long periods of time on filter paper, gauze, or any other surface im-pregnated with saline. Saline is a poor solutionfor preserving the morphology of myocardium, as it readily creates artifacts.
4. During the first six weeks after transplan-tation, at least one piece of tissue should be frozen. The working formulation recommends that the tissue be frozen in OCT compound (Miles Inc., Diagnostics Division, Elkhart, IN, USA).4We prefer to freeze the tissue using iso-pentane, which should be chilled to 2208C in a small 1.8-ml cryogenic vial. The biopsy tissue is then immersed in this prechilled isopentane cryo-vial, the cap is tightened, and the container is immersed in liquid nitrogen. At this point the tissue can be processed for immunofluores-cence or stored at 2808C for future study.
5. In the nontransplanted patient, one or more pieces of tissue can be snap-frozen for special studies (e.g., immunohistochemistry, in situ nucleic acid hybridization, polymerase chain reaction).
For transplant biopsies the working formula-tion4 recommends: “a minimum of three step levels through the paraffin block with at least three sections of each level.” Similar handling is adequate for nontransplant specimens. Slides should be stained routinely with hematoxylin and eosin; additional unstained slides should be obtained for other stains to avoid having to “face” the paraffin block again and thus minimize tissue loss due to technical handling.
For heart transplant biopsies the working for-mulation does not require routine submission of tissue from cardiac allograft biopsies for electron microscopy. However, for diagnostic “cardio-myopathy work-up” biopsies, it is important to procure at least one specimen and fix it in glutaraldehyde. If the biopsy is received in forma-lin and there are more than four biopsy pieces, one may be transferred to glutaraldehyde and submitted for electron microscopy. In cases of suspected adriamycin toxicity, consideration should be given to submitting all of the tissue for electron microscopy.