Skip to content

Optimum Specimen Handling

January 21, 2010
Stephen J. Darling, M.D.

Stephen J. Darling, M.D.

Formalin tissue fixation is accomplished by the cross-linking of tissue protein molecules which prevents the normal degradation process.  In order to optimize tissue fixation, there needs to be an adequate amount of formalin added to the specimen to assure that the specimen is completely immersed for an appropriate amount of time. Formalin is a known carcinogen and irritant and is, therefore, highly regulated.

Other fixatives, primarily alcohol based, are also available and are less toxic than formalin. These alternatives are, therefore, easier to work with and disposal is less costly.  However, none of these alternatives is as effective as formalin for maximizing antigen preservation, a quality which makes formalin ideal for most immunohistochemical stains (IHC). This is why InCyte Pathology has chosen to continue to use formalin and provides it to clients.

Paradoxically, over-fixation by formalin can mask certain antigens, which may need to be “unmasked” to improve the sensitivity of IHC. This is particularly true for estrogen/progesterone receptor studies performed by IHC as well as HER-2 assays on breast tumors. Since important therapeutic decisions are based on the results of these studies, there has been a push to standardize all phases of this testing, including fixation times. The optimum fixation time for these specimens is between 6 and 48 hours. Therefore, for breast biopsies, it is very important to designate on the requisition slip the time the specimen was placed in formalin in order for the lab to achieve this standardization.

Tissues Requiring “Special Handling”: While formalin is the preferred fixative for routine use, certain laboratory studies require fresh/fresh frozen specimens, alternative fixatives or tissue transport media. This is particularly true for the work-up of hematopoietic abnormalities requiring bone marrow and/or lymph node biopsies for suspected leukemia/lymphoma.

Multiple diagnostic modalities are often employed in the evaluation of these cases, and careful handling of these specimens is imperative to maximize diagnostic yield.  These types of studies often have stringent minimum volume requirements.  If tissue is being collected by image-guided needle sampling (rather than by open surgical biopsy), it is important to consult with the radiologist prior to the procedure to discuss the final volume of tissue that will be required.

Flow cytometric analysis is one diagnostic modality that can be very helpful in the work-up of suspected hematopoietic neoplasms. Bone marrow aspirates for this evaluation should be submitted in a well-mixed, purple top, EDTA tube. Lymph node biopsies to rule out lymphoma should be transported ASAP without fixative (“fresh”), wrapped in saline-moistened gauze or Telfa, on wet ice. Alternatively, a portion may be minced and placed into warm tissue transport media, such as RPMI, prior to transport. Specimen containers with RPMI can be obtained from InCyte Pathology upon request.

Cytogenetic studies are also becoming more important in the evaluation of hematopoietic neoplasms and certain solid tumors. The results of these studies may have diagnostic, therapeutic and prognostic significance. Bone marrow aspirates for cytogenetics should be submitted in a preservative-free, sodium heparin tube (green top).  Tissue biopsies of solid tumors, including lymph nodes, requiring cytogenetics should be transported ASAP, fresh on wet ice or in tissue transport media (RPMI).

Direct immunofluorescence testing (DIF) can be diagnostically helpful in the work-up of certain blistering diseases of the skin/mucous membranes as well as kidney biopsies obtained for the work-up of non-neoplastic renal diseases. Fresh tissue is required for DIF studies. These types of biopsies, therefore, should be transported ASAP, wrapped in saline-moistened gauze or Telfa, on wet ice. RPMI transport media may also be used for this purpose.

With the exception of kidney biopsies, electron microscopy (EM) is rarely used today as a diagnostic modality. Specimens for EM, including selected portions of a kidney biopsy,  should be placed directly into glutaraldehyde as soon as possible. These specimens should be very small, i.e., less than 1mm in diameter, since glutaraldehyde penetrates tissue very slowly. Alternatively, specimens can be placed on wet ice or into RPMI transport media and transported to the lab ASAP. In both settings, please contact the laboratory ahead of time to notify them of the need for special rapid processing.

Many of the newer molecular diagnostic tests have the most stringent specimen handling requirements. Some require that fresh tumor tissue/fluids be snap frozen within 30 minutes of removal and stored/transported at temperatures below –70 degrees Centigrade. Liquid nitrogen or dry ice slurry is used to snap freeze the specimen, which is then shipped to a reference laboratory on dry ice. Given the inherent logistical difficulties, these biopsies should only be performed in a hospital with an onsite pathologist after first consulting with the pathologist.

Conclusion: Communication with a pathologist or other laboratory personnel at InCyte Pathology prior to scheduling a biopsy potentially requiring special handling is highly recommended. This can be facilitated if the biopsy is performed at a hospital with an on-site pathologist. This will ensure proper specimen handling and avoid potential complications and the expense of needing to repeat an invasive biopsy procedure. When in doubt, simply call your local friendly InCyte pathologist for advice.


Comments are closed.