When a surgeon removes cancerous tissue, it is carefully analyzed to estimate if any cancerous cells remain in the patient's body. The “tissue margin” is the edge or border of the tissue that has been removed during surgery; specific “margins” may refer to a subset of the exterior surface. A margin is designated as “clear” when no cancer cells are found at the tissue's edge. Conversely, a “positive” result is designated when cancer cells are found at the edge of the tissue, and the implication is that not all cancer was removed during surgery. In these cases, a second surgery or some other type of clinical treatment to address the remaining cancer cells may be recommended. Tissue specimens are evaluated in the pathology lab after surgery. For some types of surgery, such as breast cancer surgery, the excised tissue specimen is also evaluated by X-ray during the operation. The clinical status of the tissue margins is considered one of the most important factors in predicting whether there will be a recurrence of the cancer. The accurate identification and reporting of the clinical status of tissue margins (i.e., “clear of cancerous cells” or “positive for cancerous cells”) is clinically relevant in a wide range of pathology specimens. Tissue marking inks are an important tool used in the determination of margin status and thereby affect the subsequent clinical action taken. Depending on the type of surgery, different colors of inks are used on a single specimen to designate: the specimen exterior surface margin; specific “margins” (or areas that are a subset of the entire specimen surface); particular anatomical features, or particular areas of concern on the specimen. Sometimes a “fixing solution” is applied after the inks to strengthen the adhesion of the ink to the tissue. Either before or after the ink is applied, the tissue is typically preserved in a “fixative” such as formalin. If the pathology analysis has a “positive” finding, the report informs the physician of the location in the patient's body where additional tissue should be removed or additional treatment directed.
A tissue specimen is often an irregular-shaped piece of tissue with fissures, crevasses or flaps on the surface. The tissue needs to be marked or labeled to designate the specimen margin(s) and original position of the tissue in the patient's body. The method of marking the tissue, which is typically to use ink, must mark accurately despite the irregularities in the surface of the specimen. If pathology analysis shows that cancerous cells are close to the exterior surface of the specimen, there may be additional cancerous cells remaining in the patient's body, requiring a subsequent surgery or other clinical treatment. For this subsequent surgery or clinical treatment to effectively address the any remaining cancer cells, it is essential that the original shape and location of the excised tissue is accurately determined. Inks that label the anatomy or the positioning of the excised tissue to indicate how it is originally positioned in the patient's body provide direction on where to target the subsequent surgery or treatment in order to more completely eradicate the cancer. Pathologists typically use inks to mark the exterior surfaces, or margins of excised tissue in cancer surgery. In some cases, the surgeon marks excised tissue before it is sent to the pathology lab.
Inks must adhere to tissue effectively for an accurate analysis in the pathology lab. They must dry quickly when applied to the tissue, both for the efficiency of use and also to maintain the fidelity of the markings without smearing or migrating on the tissue surface. The pathology lab makes slides of microscopic tissue specimens for analysis. Various materials and procedures are used to “process” the tissue specimen. The tissue is fixed, often using formalin; the pathologist then takes a slice of the tissue, typically approximately 5 microns thick, and places it on a glass slide. The slides are evaluated to identify the presence of cancerous cells, and the distance from the cancerous cells to the “margin,” or the exterior of the specimen, is measured. A wider distance indicates that the cancerous cells are less likely to have spread beyond the boundaries of the excised tissue. A shorter distance suggests that there may be cancerous cells remaining in the patient's body, and a surgical re-excision or other clinical treatment to eradicate any remaining cancer is often performed. The success of this subsequent surgery or treatment may depend upon an accurate understanding of how the specimen was originally shaped and positioned in the patient's body, or the accurate identification of important anatomical features on the specimen. The location of the cancer cells that are close to the outer surface of the specimen or on a particular anatomical feature indicate the corresponding locations where remaining cancer cells may be present in the patient. Thus, it is critical that the ink that is applied to the surface of the tissue dries quickly and adheres securely, maintaining the integrity of the marking, without running, dripping or migrating to an adjacent area on the surface of the specimen or into a fissure or crevasse. If the ink does not dry quickly the fidelity of the markings are affected and the ink may smear or migrate on the tissue surface. If the ink migrates to an adjacent area on the surface of the tissue, and that area is determined to have cancerous cells close to the surface, then the surgical re-excision or clinical treatment directed at remaining cells in the patient may target the wrong location, resulting in untreated cancer cells which may cause a recurrence of the cancer. The most serious negative outcome is a local recurrence of the cancer. Of the 4% to 20% of lumpectomy patients who suffer a recurrence of their breast cancer, half of these cases are metastatic, or potentially deadly. If the ink migrates into a fissure or crevasse in the tissue, the pathology report may result in a “false positive,” because the cancerous cells appear closer to the exterior surface of the tissue specimen than they actually were. A false positive could result in unnecessary surgery or clinical treatment for the patient. In breast cancer cases, re-excisions occur for up to 60% of lumpectomy patients, and of these, as many as 66% are false positives.
The viscosity of commercially available inks is widely variable. Thin inks often run and drip when applied to tissue, which prevents the accurate designation of specific tissue margins, which is the purpose for inking tissue. Moreover, thin inks have a great percentage of water and take a long time to dry. Thin inks can also run into crevasses of tissue, whereby the ink becomes closer to the cancer cells in the specimen, causing the margins to be misinterpreted, and creating a false positive result that may cause an unnecessary second surgery or treatment. On the other hand, thick inks can be lumpy and sometimes contain dry chards of crystallized matter, which appear to be microcalcifications (potentially cancerous cells) on the X-ray image taken during the surgical procedure. These dry fragments of ink may cause false positives in the interpretation of the X-ray image, resulting in unnecessary removal of additional tissue, or at a minimum causing confusion and delay during the operation while the patient is anesthetized.
Adherence to tissue can be problematic with commercially available inks when these inks are applied to tissue and the tissue is fixed in formalin. Tissue may be fixed in formalin before or after ink is applied. If tissue is fixed in formalin first, then the inks often adhere less effectively to the tissue. Commercially available inks do not adhere well to tissue that has been pre-fixed in formalin. If the inks are applied first and then the inked specimen is submerged into formalin, the inks may wash off of the tissue and into the formalin.
Commercially available inks are not sterile, which also results in problems. First, non-sterile inks may allow cross-contamination among tissue specimens because the applicator used to apply the ink may touch the specimen and then touch the bottle during the process of applying ink. Second, if the surgeon applies non-sterile inks while in the operating room, the non-sterile inks may not be used in the sterile field, which introduces the risk of errors if the specimen is carried across the room to the non-sterile area before it is inked or if inking is delayed until the surgery is complete. Third, non-sterile inks sometimes feature dry fragments that look like microcalcifications, or potentially cancerous cells, on the intraoperative X-ray image.
Other problems exist with commercially available inks. Although accurate identification of ink color is essential to effective clinical usage of the inks, the colors of commercially available inks are often difficult to distinguish from one another. To perform effectively, each ink color must be both recognizable and distinguishable from other colors under both reflective light (ordinary lighting conditions, as when the ink is applied to tissue in the pathology lab or operating room), and under transmitted light (when light shines through from the opposite side, as when the tissue on a slide is placed under a microscope). When the inks are viewed under reflective light, dark colors such as blue, violet and green are often difficult to distinguish from one another and all appear to be black. When applied to tissue and viewed on the slides under a microscope using transmitted light, difficulties are often encountered with the yellow-orange, orange-red, blue-violet and red-violet comparisons. Confusion accurately identifying the ink color on the specimen can lead to erroneous interpretation regarding the location of cells that are close to the specimen surface, resulting in re-excision or treatment in an incorrect location, and possibly cancer recurrence.
When inks are applied to tissue specimens, a fixing solution is sometimes used. The purpose of the fixing solution is to enhance the adherence of the inks to the tissue. However, commercially available fixing solutions are problematic in that they fail to effectively strengthen the adherence of the inks to the tissue, resulting in ink washing off when tissue is pre-treated with formalin before inking, submerged in formalin after ink is applied, or when a knife is used to cut through the inked specimen. In addition, some fixing solutions have an odor that is offensive to clinical staff.
Accordingly, there is a need for colored inks for marking tissue specimens that overcome the foregoing problems of commercially available inks. There is also a need for a fixing solution that prevents the wash-off of inks from the tissue specimens when the tissue is exposed to formalin before or after ink is applied.