Biopsy Information and Courses from MediaLab, Inc.

Several types of GYN specimens are routinely submitted to clinical histology laboratories such as cervical loop electro-cautery excision (LEEP) procedures, cervical cone, endometrial curettage, and vulvar biopsies. Although for most GYN specimens you will follow the same general guidelines as for other similarly shaped and sized tissue specimens, it is especially helpful to understand a little about the surgical procedures used to obtain these specimens and also the common methods for dissection used.Cone biopsiesThis procedure is a conical excision of the cervical canal using a laser or cold blade.The wider part of the cone is the ectocervix, and the tapered tip contains the endocervical margin.The ectocervix by convention is described as a clock face, with the most superior midpoint of the anterior lip designated at 12 o'clock.Sections will usually be submitted sequentially and designated in the gross description by their clock face orientation.These sections should be embedded so that perpendicular sections will be shown of the cut surface.Cervical LEEPThese related procedures remove less tissue than a cone biopsy and are obtained by electro-cautery of the cervical transformation zone.The specimen may be divided either perpendicularly or by using a radial dissection method.Endometrial curettageCurettage is a scraping method, in this case of the endometrial lining.The specimen obtained will consists of bloody fragments.These may be submitted in biopsy bags to contain the fragments during tissue processing.The surface of the bags or paper should be scraped lightly to remove as much material as possible.Embed to keep the fragments centralized in the block face and to arrange for the greatest surface area to be shown in one plane.Wipe forceps and all surfaces well after these specimens; it is easy to transfer the loose bloody fragments to other specimens.Embed all fragments with respect to the ink present with inked edges facing all in the same direction.

The punch or trephine biopsy is one of the most popular methods of sampling for diagnosis of inflammatory skin conditions. A special instrument is used to cut out a cylindrical shaped piece of skin. The skin core contains the full skin thickness with the main skin layers (epidermis, dermis, and subcutaneous) usually being present. The punch biopsy must be embedded so that sections are taken perpendicular to the epidermis. This typically means that the specimen will be laid on its side (it is rounded and has no clear edges), with the epidermis disc being clearly visible on one side. If the punch biopsy is larger than 0.4 cm in diameter, it may be bisected. In bisected punch biopsies, each half will be laid on its side (cut surface) into the block face.

The following steps can be taken if you open a cassette and it appears that NO specimen is within:Look carefully in the cassette lid and interior corners and crevices. If you still find no visible tissue, ALWAYS have a second (or even third person) verify and help you check for any tiny fragments that you might have missed.Check the gross description, it may indicate that the specimen was very minute and was not expected to survive processing. If the specimen has been submitted in lens paper or a mesh biopsy bag, scrape all surfaces with a warmed, dull knife and transfer ANY, perhaps unseen, cells or particles to a mold where you have placed a small amount of molten paraffin. Anything microscopic that might be present will be likely to "float" off the knife, and can then be transferred to the block where it may be visible in the final section. Retain the lens paper or mesh bag in the cassette lid to document that "no tissue was seen."Individual laboratory procedure should be followed when documenting specimen loss on the accession log and/or laboratory information system (LIS).Missing fragments (less than dictated) that have been inadvertently dropped, can sometimes be retrieved, salvaged, and identified by retaining cassette lids in a separate bag for 24 to 48 hours (or until the case is signed out).

Bone marrow aspiration and biopsy are standard tools used in the hematology laboratory to aid in the evaluation and diagnosis of peripheral blood abnormalities. Some of these abnormalities include: cytopenias (such as neutropenia), thrombocytopenia and anemias. Bone marrow aspiration and biopsies are also used by hematology/oncology specialists in the diagnosis of leukemias, dysplastic syndromes, and proliferative syndromes. A bone marrow aspiration and biopsy may also be part of the evaluation of fever of unknown origin (FUO), failure to thrive(FTT) in the pediatric setting, as well as some metabolic and genetic disorders.A bone marrow aspirate sample is obtained by inserting a needle into the bone marrow space and withdrawing 5- 10 milliliters (mL) of marrow in several different syringes. These samples are then transferred to evacuated blood collection tubes containing the anticoagulants required for the types of assays desired. A portion of this liquid marrow is smeared for staining and evaluation under light microscopy. It can be sent for various types of laboratory assessment including : immunophenotyping, cytogenetic evaluation, and molecular analysis.While bone marrow aspirations and biopsies are usually obtained by the hematologist or oncologist, they are evaluated and interpreted by a hematopathologist with the assistance of the laboratory technologists who prepare and stain the smears. In many laboratory settings the technologists also perform the bone marrow differentials.

Some pathologists prefer their bone marrow smears to be made fresh at bedside without the use of any anticoagulants. This however limits the number of smears that can be made before the sample clots. Using a syringe, that has been rinsed with preservative-free heparin, to pull the marrow during the procedure will prevent clotting but will introduce morphology changes and staining artifact.It is preferable to make smears as soon as possible after sample collection. However, when stored in the refrigerator, acceptable smears can be made from an EDTA tube as long as 8-10 hours after sample aspiration without introducing excessive amounts of artifacts. This is useful when marrows are collected at times when staffing trained in marrow smear preparation may not be available.

Depending on the laboratory protocols and the hematopathologist's preference, the technologist's responsibilities in bone marrow aspirate processing and reviewing can vary from simply staining slides to complex tasks such as smearing, staining, counting and distributing samples based on laboratory standard protocols. While laboratory professionals may perform bone marrow differentials, it is the hematopathologist's responsibility to review, interpret, and verify those counts after evaluating all of the samples that were provided. This includes both the bone marrow aspirate and biopsy smears, as well as the flow cytometry data, if required. When differentiating and counting samples with increased numbers of blasts, the technologist may make a determination of cell line for the blasts, according to observations on a Wright or Wright-Giemsa stained slide, but the hematopathologist may modify this placement based on flow cytometry data and additional special stains.The laboratory technologist can assess cellularity, presence of megakaryocytes, and possible presence of tumor cells on the bone marrow smears. Ultimately, the hematopatholgist is responsible for interpreting the bone marrow differential results after meticulous review of all smears stained, as well as any biopsy sections available. It is also the hematopatholgist's responsibility to identify tumor cells that may be present.In laboratories that use a standard order set with sample drop off directly from bedside, it is the technologist's responsibility to split and distribute the marrow based on laboratory protocol. It is hematopathologist's responsibility to verify that standard order testing was actually sent and to add on any additional testing deemed necessary, based on clinical history and lab findings. Once you become comfortable with bone marrow morphology and more familiar with your laboratory's protocols, performing marrow differentials will become less intimidating and more of a collaboration between technologist and hematopathologist.

While smears from the bone marrow aspirate are the most common preparations, touch preparations (touch preps) made from the bone marrow biopsy core may also be useful or necessary. When aspirates are difficult or a dry tap occurs, the only sample available to be evaluated in the hematology laboratory may be the bone marrow biopsy. To create a fresh biopsy touch preparation, the fresh bone marrow core is gently rolled between two slides, then gently rolled between five or six pairs of coverslips. There should be enough cellular elements present when using this method for the laboratory professional to evaluate. The imprints will be wet and cellular at first but as the surface dries it will eventually become less cellular. At this point the core is placed in fixative and sent to pathology for evaluation. The number of touch preps you can make is dependent on how wet/ bloody or fibrotic the core is to begin with, but even one set can be enough to aid in diagnosis.While it is not practical to practice making touch preps from a real biopsy core, it is possible to practice the technique by using a length of applicator stick soaked in either blood or stain to simulate a real biopsy core. To do this, simply break off a short, 0.5 inch piece of a standard plain or cotton tipped applicator stick and soak it in the fluid of your choice. As you roll it between slides or coverslips you will see the pattern it leaves behind. Think of the motion of a teeter-totter (seesaw) as you roll. There should be very little downward force on the core as you coax it to roll. If the core will not roll then you can just touch the slides or coverslips to the surface of the core few times on each slide.Note: If the biopsy is placed directly into fixative and sent to pathology, it must first be decalcified before it can be sectioned and stained. This process will take at least 24 hours depending on the lab and if additional stains are required, it may be at least 48 hours before a result is released.

When the technologist accompanies the clinician to assist with the bone marrow aspiration procedure to make smears at the bedside, it is necessary to understand the role of the clinician and the technologist.The clinician is responsible for patient positioning and sterile preparation, pain control, and performing the aspirate and biopsy. The clinician often hands off sample syringes to the technologist, once collected. The clinicians are responsible for providing the procedure kit and fixative for the biopsy, all labels, and obtaining the requisitions and a copy of the clinical history for the hematopathologist. The technologist will set up a mini workspace near the bedside where the samples are split into the required tubes. Smears are then prepared from the aspirate as well as biopsy touchpreps before the biopsy is placed in fixative. In this setting the technologist will usually deliver the samples and requisitions to pathology and continue the processing procedure.The kit the technologist brings to the bedside usually contains mini petri dishes, coverslips, slides, microcapilary tubes or Pasteur pipettes, micro-pipette bulb and the various evacuated blood collection tubes and media flasks required for the standard bone marrow draw.Most institutions will have a standard draw and testing protocol designed to ensure that enough sample is obtained to cover all of the usual testing requirements. An example would be a three-syringe-draw with the first two syringes containing no anticoagulant and the third syringe rinsed with preservative-free heparin. The first dry pull would be split between a green and a purple top evacuated blood collection tube and would be used for morphology (EDTA) and flow cytometry and cytogenetics (green) if needed. The second dry pull is split into two additional purple top tubes plus a green top tube and would be used for molecular assays such as SNP array, Flt-3, JAK2, MPL mutation, etc. The final heparinized syringe could be used for other treatment protocol requirements or to provide sample for additional assays.

The macrophage is the final stage of development in the monocyte lineage. It is a phagocyte whose roles include the removal of dead and dying tissue and the destruction and ingestion of invading organisms. Macrophages (histiocytes) act as immune modulators as they will present antigens from ingested pathogens to helper T-cells.Their primary role in the bone marrow is the removal of cellular debris, including old red blood cells (RBCs). As a result, they become a source of iron for maturing RBC precursors. A histiocyte is a less phagocytic form of a macrophage with fewer lysosomal granules. Histiocytes may form clusters, or even fuse together into mulitnucleated giant cells. These giant cells are particularly evident on bone marrow biopsy from a patient with a marrow granuloma.The top image on the right shows the early transformation of a monocyte into a macrophage (see red arrow). Notice the increase in the amount of cytoplasm present as the cell begins to ingest debris in the bone marrow. This is demonstrated by the increasing vacuolization present in the cytoplasm. The larger the debris ingested, the larger the vacuoles will be.The lower image on the right shows a macrophage with large vacuoles (red arrow) adjacent to an RBC cluster (blue arrow). This is a common placement, since the macrophage is the iron source for these developing RBCs in the bone marrow.

The prototype history for this organism is either a still birth or a neonate with death ensuing within two or three days post-partum due to high fever, sepsis, and respiratory distress. The mother usually experienced a flu-like illness late in the third trimester of pregnancy, characterized by low-grade fever, myalgias, malaise, and backache. In this case, biopsy material of brain tissue obtained at autopsy was submitted to the pathology laboratory for tissue diagnosis and fluid from the pia-arachnoid was sent to the microbiology laboratory for culture.

The H & E section of the brain biopsy (left frame of image)revealed edema of the parencymya with the accumulation of inflammatory cells in the perivascular spaces. The close in view of the exudate (right frame of image) reveals that the inflammatory exudate is comprised primarily of polymorphonuclear luekocytes. The histologic diagnosis therefore is suppurative meningitis, with culture results necessary to establish the etiologic agent.

In addition to hereditary hemochromatosis (HH), there are other conditions of iron overload that must be considered in a differential diagnosis. Disorders such as sickle cell disease, thalassemia, sideroblastic anemia, congenital dyserythropoietic anemia, and liver disease may also cause iron overload. Transfusion-dependant patients and persons who abuse iron-containing vitamin supplements are also at risk. These conditions are usually described as secondary iron overload, in contrast to the primary iron overload of HH.Patient history, clinical signs and symptoms, biochemical and hematologic laboratory analyses, and possibly results of a liver biopsy may be needed to establish a diagnosis of a condition causing secondary iron overload. DNA tests for common HFE mutations are very likely the most important diagnostic tool for identifying HH as the cause of iron overload. In some patients, both secondary causes and HH may be contributing to iron overload. Differentiating the secondary causes of iron overload from HH is heavily dependent on the results of laboratory assays, but a complete discussion is beyond the scope of this course.

Cervical cytology has been quite successful in the detection of cervical cancer. Since the carcinogenesis of cervical cancer is usually very slow, taking many years to decades to develop, cervical cytology can detect HPV-related cervical cancerous or precancerous states in most cases. The Papanicolaou-stained smear (Pap smear), is used most often to screen for pre-cancerous and cancerous processes by looking for changes to normal cervical cells. Abnormal cytology findings are investigated with colposcopy and biopsy. Colposcopy utilizes a powerful light source and lenses to locate and identify lesions that led to abnormal cells on Pap smear slides. If lesions are found, they too can be biopsied during colposcopy. For a Pap test, cervical cells are collected, fixed on a glass slide or placed in a liquid medium, fixed on slides, stained, and microscopically examined for abnormal cells. Sensitivity of a Pap smear is only 55%-80%; however its specificity is greater than 90%. Further information regarding cell types and terminology associated with the Pap test/cervical cytology testing are provided in the PDF resource that can be accessed from this page.

The biopsy specimen is usually fixed in Zenkers' acetic solution (5% glacial acetic acid; 95% Zenkers) for 6-18 hours or B-5 fixative for one to two hours. Excessive time in either fixative makes the tissue brittle, then briefly decalcified. The tissue is processed together with other tissues, and is embedded in paraffin and cut at 4 µm and stained with hematoxylin and eosin (H&E) and Perls' Prussian blue iron stain, and other special stains as indicated.

A bone marrow biopsy involves removing a small portion of the bone marrow without destroying the architecture of the marrow. This type of biopsy is necessary when the marrow cannot be aspirated (dry tap) due to a disease process, and also provides additional information complementary to that derived from the aspirate: biopsy specimens are more accurate for assessing cellularity, and infiltrative processes, such as metastatic carcinoma, fibrosis, amyloid, and lymphoma. A biopsy specimen is processed as follows: touch preparation tissue section

This low power view is from a biopsy of a patient with normal iron stores. Note the presence of iron shown by the arrow.

Markely increased stainable iron is present in this biopsy. Iron stores may be increased in sideroblastic anemia, chronic infections, hemochromatosis, hemosiderosis due to numerous blood transfusions, chronic hepatitis, cirrhosis, and uremia.

The microscopic examination of marrow smears can be divided into three main steps.Evaluating cellularity from the biopsy/particle smearEvaluating marrow iron from the biopsy/particle smearMorphology examination from the Romanwsky stained smears

This biopsy section was taken from a patient who has very few cellular elements in the marrow. Notice that over 90% of the marrow is composed of fat. If all of the cellular elements are decreased, the patient's condition is said to be pancytopenic or aplastic. There are numerous causes for aplasia, including drugs such as chloramphenicol, chemotherapy and inheritance (Fanconi's Anemia).

This is another view of the same slide showing increased cellularity and decreased fat.

The setting is the cafeteria in a hospital or the lounge in an independent laboratory. Two employees, a billing clerk and a medical technologist, from different departments are having lunch together. The billing clerk mentions that she saw a bill go through the system for one of her coworkers for a biopsy. She asks the medical technologist if she has the necessary security level access to see pathology test results because she is concerned about the welfare of the coworker. The medical technologist does have the necessary security clearance to see the results. She should:Correct Answer: Refuse to look up the results for the clerk and remind the clerk that it is a violation of compliance policies to do so, or to ask another to do so. Discussion: The medical technologist has a responsibility to report violations of compliance policies and the friend has put her in a difficult position. For that reason, it is not enough to just refuse the clerk's request. If the medical technologist does not take the responsibility to inform the employee of the policy then there is a possibility that the employee would ask some other employee to do it for her.

The setting is the cafeteria in a hospital or the lounge in an independent laboratory. Two employees from different departments are old friends are having lunch together. A billing clerk and a medical technologist are friends and are having lunch together. The billing clerk mentions that she saw a bill go through the system for one of her coworkers for a biopsy. She asks the medical technologist if she has the necessary security level access to see pathology test results because she is concerned about the welfare of the coworker. The medical technologist does have the necessary security clearance to see the results. She should:Correct Answer: Refuse to look up the results for the clerk and remind the clerk that it is a violation of compliance policies to do so, or to ask another to do so. Remind her of the requirement for each employee to report any violations of policy. Discussion: The Medical technologist has a responsibility to report violations of compliance policies and the friend has put her in a difficult position. For that reason, it is not enough to just refuse the clerk's request. If the medical technologist does not take the responsibility to inform the employee of the policy then there is a possibility that the employee would ask some other employee to do it for her.

Clinical specimens where organism may be encountered: CSF Blood Stool (rare) Vesicle fluid, skin swab, or biopsy Gram stain morphology from clinical specimens: Large, gram-positive rods with square or concave ends in short chains Spores are usually NOT present Capsule may be viewed in smears from infected tissue, but this is NOT reliable Gram stain morphology from culture material: Large, gram-positive rods with square or concave ends, often in long chains (more than 2-4 cells) Cells easily decolorize as the culture ages Does NOT form capsules in culture Central to sub-terminal, oval spores, with NO significant swelling of the cell It must be noted that spore production increases with the age of the culture. Do NOT keep these cultures in the laboratory for longer than 24 hours for this reason!

This is a high power image of a liver biopsy. Notice the nuclear detail within the hepatocytes or liver cells. There are red blood cells sitting within the sinusoids, which are types of blood vessels.