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Biopsy Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Biopsy and links to relevant pages within the course.

Learn more about laboratory continuing education for medical technologists to earn CE credit for AMT, ASCP, NCA, and state license renewal and recertification. Or get information about laboratory safety and compliance courses that deliver cost-effective OSHA safety training and continuing education to your laboratory's employees.



Basic Tissue Orientation and Paraffin Embedding Technique
Gynecological (GYN) Specimens

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.

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Skin Specimens

Specimens of skin will be submitted to histology after being obtained by various surgical biopsy procedures, such as the shave, punch, or curettage methods. In general, skin specimens will be oriented so that sections will be taken at an angle perpendicular to the epidermis. Identification of the epidermis is a good first step in deciding correct orientation of most skin specimens. However, since important diagnostic information can very easily be lost with incorrect orientation of skin samples, it is important to verify the specimen type and biopsy type using the accession log, embedding worksheet, or gross description before proceeding. Do NOT merely rely on visual recognition by itself.

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Punch (Trephine) Biopsies

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.

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A small tubular artery is received in histology to rule out temporal arteritis. When embedding this specimen, how must the sections of the tube must be oriented?View Page
On all of the following specimens, the histologist is given the embedding orientation instructions to "embed on edge." For which specimen is this orientation NOT appropriate?View Page
Identify the tissue type shown in the cut surface of this paraffin tissue block.View Page
Practices for Specimen Loss

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).

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Bone Marrow Aspiration Part I: Normal Hematopoiesis and Basic Interpretive Procedures
Introduction to Bone Marrow Aspirates and Biopsies

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.

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Clinical Laboratory's Role: Bone Marrow Aspirates and Biopsies

While the role of the hematopathologist in the interpretation of bone marrow samples is well defined, the role of the technologist can vary greatly depending on the laboratory and the clinical setting.In some cases, physicians deliver prepared bone marrow smears to the laboratory that they have been prepared at the patient's bedside . In other settings, the technologist assists in the bone marrow collection procedure by making bone marrow smears at the bedside. There are also situations where a physician will bring anticoagulated bone marrow in specimen tubes to the laboratory for the technologist to smear, stain, and distribute as the hematopathologist requires.Once the marrow smears are prepared and stained, the next steps will vary depending on the laboratory. In some institutions it may be the hematology or oncology fellows/attending physicians who are responsible for counting and evaluating the aspirate smears, while the biopsy samples go to pathology. In other settings it may be the technologists who perform the differentials while hematopathology completes the evaluation and interpretation.

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In which of the following situations would a bone marrow aspirate and biopsy possibly be used as an aid for diagnosis?(Select all that apply)View Page
Bone Marrow Biopsy

A bone marrow biopsy is obtained by a similar technique but rather than aspirating liquid marrow, a core of bone is allowed to enter the biopsy needle and is then removed within the needle. The image* illustrates the body site where the bone marrow is normally obtained.The core can be rolled between two slides to make touch preparatons (touch preps), which are then stained and reviewed. The bony core is fixed and sent to pathology, where it will be decalcified and prepared for sectioning and staining.Reference: Bone marrow biopsy. National Cancer Institute. Available at: http://www.cancer.gov/Common/PopUps/popDefinition.aspx?id=46506&version=Patient&language=English. Accessed July 3, 2011.

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Bone Marrow Aspirate and Biopsy Collection

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.

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Rules for Bone Marrow Differentials

Bone marrow differentials have significant differences from peripheral blood differentials that need to be considered as they are reviewed and counted.One of the most important facts to consider is the large variability in cellularity and cell distribution depending on the type of preparation that is used. Choosing where to count and when to use which of the smear types available to you, takes time and experience and can be directed by a pathologist's preference.Regardless of how many, or what types of smears you have available to choose from, you will always start with a simple visual inspection of your smears. Begin by recording the patient identification information as well as date of sample, and any other mandatory patient identifying information necessary for your laboratory. Record aspiration site information when provided. Many patients will have bilateral bone marrow aspirates performed as part of a diagnostic or staging workup. Standard aspiration sites are: posterior iliac crest (PIC), anterior iliac Crest (ANT), sternum (S), spinous process (SP) and sometimes in very young children, bone marrow is obtained from the tibia (T). Be aware, that while a bilateral bone marrow aspirate usually involves an aspirate of the same site from opposite sides of the body, e.g., L-PIC and R-PIC, in some situations, a bilateral staging aspirate will be from two different compartments on the same side, e.g. R-AIC, R-PIC. Observe the appearance of the bone marrow smears. Do any have feather edges? Are there fragments or spicules present on any of the smears available? If so, they should be your first choice to view, since they are more representative of what the biopsy will show if one was obtained. Once you select your smears, scan using 10X magnification on the microscope. Are some of the fragments/smears so thick that you cannot see good morphology? If so, reject these areas/slides. Are some of the fragments/smears so thin that everything is smashed? These areas/smears cannot be used either. Are there areas in the vicinity of any of the fragments that have good staining characteristics as well as readable morphology? This is where you should begin your differential.

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Responsibility of the Technologist versus Hematopathologist

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.

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Long Slide Preparation Techniques: Wedge Smear

The simplest way to prepare a bone marrow smear is to treat it similarly to a peripheral blood smear. A drop of well-mixed marrow sample is placed near one end of slide and a second slide is used to draw it the length of the slide until a feathered edge is produced. The slide should be air dried and labeled with the patients identification, date of collection, sample site or lab accession number (depending on the laboratory's standards).If the marrow is rich in spicules or fragments, there will be "clumps" of marrow fragments at the end of the feathered edge where these fragments are deposited. Fragments, also known as spicules, are aggregates of bone marrow cells that are pulled from the bony matrix of the marrow space (trabecula) during the aspiration process. They represent what will be seen on the bone marrow biopsy between the bony trabecula, once the biopsy is decalcified and sectioned. Since these spicules are cohesive aggregates of cells, they will not spread well if a wedge smear technique is used, which may make it difficult to identify cell types present in the thicker parts of the spicules.Another disadvantage of using the wedge technique to make smears of bone marrow is the fact that the distribution of cells will not be uniform or representative of how the cells are distributed in the marrow. This technique tends to skew the spread, based on the size of the cells present. Just as the larger bone marrow spicules wind up at the end of the feathered edge, so do cells like megakaryocytes and other aggregates, such as tumor clumps. However, since there tends to be more of a feathered edge in this type of preparation, it can be preferable to other methods when the marrow to be evaluated has no fragments. Long slide prepared smears are particularly useful when evaluating leukemic patients at days 8 and 15 of therapy, when the cellularity is greatly reduced and clear morphology is needed to differentiate between treated lymphocytes and treated blasts. In this case, it is easier to distinguish blasts from lymphocytes when this smear technique is used.

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Biopsy Touch Preparation Technique

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.

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Bone marrow Smear Preparation

Accurate interpretation of bone marrow aspirate specimens is highly dependent on two factors: The quality of the sample obtained by the clinician The quality of the prepared bone marrow smears While the technologists have no control over the quality of the aspirate obtained by the clinician, in most cases they are able to impact the quality of laboratory prepared smears.The method used for preparing slides may be dependent on sample volume, staining techniques, storage space, clinical setting, patient status, and hematopathologist's preference. Bone marrow sample preparations can be made on slides or coverslips from a direct marrow sample or a sample that is manipulated to enhance cellularity (selected for marrow fragments or spicules). In situations where the technologist goes to the bedside, touch-preparation (touch-prep) smears from the biopsy core can be prepared before it is placed in fixative.Regardless of which method is the preferred primary technique at your institution, it is useful to be familiar with other methods, since there may be a situation when the use of an alternate method is the only way to obtain an interpretable smear.When adequate samples are provided, it is desirable to make several slides or coverslips so that there will be enough preps available for all the possible tests/special stains that may be requested. The extra smears can be stored at room temperature and protected from light in an envelope or sleeve for further testing, if required.

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Which of the following smear techniques can be utilized when processing bone marrow aspirate samples? (Choose all that apply)View Page
Iron Staining

Iron staining on bone marrow aspirate smears is commonly part of the standard order protocol for bone marrows aspirates. The iron staining procedure utilizes the Prussian Blue stain for ferric iron to assess bone marrow iron stores. This procedure is particularly helpful when evaluating patients with anemia, iron overload, myelodysplasia, etc. In the adult setting, it is commonly performed on the bone marrow biopsy, but can be requested on the aspirates as well.In the pediatric setting, it is less likely to be part of the standard order set since young children rarely have stainable iron stores. However, iron staining may be requested on patients with congenital anemia and possible mitochondrial defects to look for sideroblastic anemia.In this technique, iron will stain blue and will normally be found in bone marrow stromal/ macrophages, which are found in the spicules. On aspirate smears, without fragments/spicules, it is not possible to evaluate for iron stores. However, if there are nucleated red blood cells (NRBCs) present, it is still possible to look for the ringed sideroblasts, common in sideroblastic anemias.The image on the right is a field from a bone marrow slide from a patient with congenital sideroblastic anemia. The NRBC indicated by the red arrow is a normal siderocyte with few granules of hemosiderin scattered through the cytoplasm. The NRBC that is indicated by the blue arrow has a large number of hemosiderin granules concentrated in the mitochondria that surround the nucleus. This is a pathologic ringed sideroblast.

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Bone Marrow Collection: Patient Bedside

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.

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The role of the laboratory technologist in processing bone marrow aspirates can vary depending on laboratory and clinician protocols. Which of the following roles may be performed by a laboratory technologist?(Select all that apply) View Page
Macrophage (Histiocyte)

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.

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Case Studies in Clinical Microbiology
Clinical History

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.

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A Brown and Brenn gram stain was performed on one of the tissue biopsy specimens. Organisms were seen as shown in the image. Based on the history and the appearance of the bacteria, the most likely identification is:View Page
Histology of Brain Biopsy

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.

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Hematology / Hemostasis Question Bank - Review Mode (no CE)
What is the site used most frequently in the adult patient when performing a bone marrow biopsy:View Page
What is the normal cellularity of an iliac crest bone marrow biopsy in a 50 year old male:View Page

Hereditary Hemochromatosis
Secondary Disorders of Iron Overload

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.

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Definitive Tests for Iron Overload

Measuring the amount of iron deposited in the liver is considered definitive for iron overload. This may be done by liver biopsy, computed tomography (CT), or magnetic resonance imaging (MRI). Demonstrating iron in parenchymal liver cells helps determine disease severity. Liver sections obtained by biopsy are stained with Perls Prussian blue which stains iron present in parenchymal cells. A photomicrograph of this reaction is shown.Although liver biopsy may not be necessary for diagnosing hereditary hemochromatosis (HH), it offers the advantage of detecting liver fibrosis if present. Molecular tests for mutations associated with HH are considered the gold standard of current HH testing. Liver biopsy is not needed for diagnosing all patients suspected of having HH, but may be ordered in some cases.

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Which statement best describes the use of the liver biopsy in suspected cases of hereditary hemochromatosis (HH)?View Page
Quantitative Phlebotomy

An alternative to liver biopsy as a means of documenting iron overload may be provided by quantitative phlebotomy performed during treatment. The removal of 4 to 5 grams of iron through documented successive phlebotomies (16 to 20 phleblotomies) without development of anemia is indicative of iron overload. (One unit, or 450 mL, of blood is assumed to contain approximately 200 to 250 mg of iron.) Quantitative phlebotomy is useful in patients for whom liver biopsy is contraindicated or patients who refuse the procedure.

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Human Papillomavirus (HPV) and Molecular Diagnostic Testing
Pap Smear

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.

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Pap Smear Limitations

The Pap test is not a diagnostic test; it is only a screening test for cervical cancer or cervical changes that are precancerous. For cervical cancer diagnosis, colposcopy, biopsy and/or endocervical scraping are needed. The low sensitivity associated with the Pap smear test is another limitation and involves factors such as: inadequate collection, sample preparation errors, and the possibility of inaccurate microscopic examination (although numerous safeguards are built into the process in an attempt to prevent inaccuracies).

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Introduction to Bone Marrow
Preparations which can be made from the bone marrow aspiration specimen include:View Page
Which of the following statements are FALSE regarding the bone marrow BIOPSY specimen?View Page
A bone marrow biopsy from a 50-year-old patient that has an overall cellularity of 20% (i.e., 80% fat and 20% hematopoietic cells) is considered:View Page
Collection of the Aspirate

The marrow aspiration is usually performed before a biopsy is done. A syringe is attached to the needle, the plunger is pulled and 1.0-1.5 ml. of marrow particles and blood from marrow sinuses is withdrawn. If additional bone marrow samples are needed, a separate syringe must be used each time. If more than 2 cc. per syringe is taken out, the blood to marrow ratio will be too high and the preparations will not accurately reflect the marrow contents. As the marrow is aspirated into the syringe the patient will feel some pain and pressure even though local anesthetic has been administered.

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Fixation and Processing of the Biopsy Specimen

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.

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Advantages of a Biopsy Specimen

Examining the biopsy allows the structure of the marrow to be viewed as it exists in the body. It provides essential diagnostic information in conditions that disrupt the normal architecture, such as metastatic carcinoma, myelofibrosis, Hodgkin's lymphoma and granuloma. A biopsy may also be used to evaluate cellularity and identify acid-fast bacteria or fungi in less time than is needed for routine culture methods. One disadvantage of the tissue sections prepared from the biopsy sample is that morphologic detail is lost. For this reason, in many cases imprint slides or smears from the aspirated sample are also examined.

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The most common site for bone marrow biopsy and aspiration in adults is:View Page
Match the phrase with its description.View Page
Which of the following statements are TRUE regarding biopsy specimens? (Choose ALL of the correct answers)View Page
Preparation of Direct Smears

The sample in the first syringe is quickly delivered into a watchglass or onto a slide. After the technologist verifies the presence of white-gray marrow particles in the sample, push smears and/or coverslip smears from this unanticoagulated sample are made immediately. All films should be rapidly air dried. The appearance of fat as irregular holes in the films also give the assurance that marrow and not just blood has been obtained. This type of smear is referred to as a direct smear and is usually used to evaluate morphology. Although some evaluation of cellularity and M:E ratio is possible, particle smears or biopsy sections provide a more accurate representation of these factors.

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Collection of Bone Marrow Biopsy

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

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Biopsy Touch Preparation

A touch prep is made by holding the biopsy plug with a forceps and touching the plug to one or more clean slides in several places. Imprints of the marrow remain on the slide. The slides are quickly air dried, fixed with methanol and stained with Wright-Giemsa or other cytochemical stains. Morphologic details of the cells are preserved with this type of preparation.

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Low Power View of Prussian Blue Stained Biopsy

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

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Another Biopsy Showing Normal Iron Stores

Another view of a biopsy showing normal amounts of iron.

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Increase Marrow Iron Stores

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.

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Biopsy Section and Bone Marrow Smear

The biopsy section and bone marrow smear can both be used for evaluating iron stores. If the biopsy section is used, the fixative chosen for processing the specimen should not contain acid. Acid fixative can remove iron from the tissue, producing the false impression of iron deficiency.

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Microscopic Evaluation of Marrow Smears

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

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Medium Power View of Biopsy

This biopsy also has a cellularity of 25% and approximately 75% fat, and is therefore slightly hypocellular.

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The peripheral blood platelet count in this patient will likely be:View Page
Evaluating Cellularity

The biopsy section or particle smears are the preparations that are preferred for the evaluation of marrow cellularity and architecture. The low power objective is used to examine the slide and compare the cellular area to the amount of fat (fat cells appear as white circles interspersed among the cellular elements). On the biopsy section the specific type of cells present are difficult to determine but the cellularity can be clearly seen. The particle smear may be used to evaluate cellularity as well as morphology. The diagnostic significance of the evaluation of cellularity, is simply to see if there are too few, too many, or sufficient cell precursors present in the bone marrow. A general estimate of the expected normal cellularity range in an adult can be determined by first subtracting the age of the patient from 100%. The range is then +/- 10 of that number, e.g., a normal, healthy 70-year-old adult should have an overall cellularity between 20% and 40%.

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Hypocellular Bone Marrow Biopsy

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).

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Low Power View of Biopsy

This low power view of a hematoxyln and eosin stained bone marrow biopsy shows fat cells as clear circles, and the darker intervening areas as blood cell precursors. This biopsy is about 25% cellular, or mildly hypocellular. A normal marrow in a middle aged adult is about 50% cellular.

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Hypercellular Bone Marrow Biopsy

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

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Hypercellular Bone Marrow Biopsy

A low power view of a biopsy section stained with hematoxylin eosin stain. This section is showing increased cellularity and decreased fat. This specimen is about 85% cellular.

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Medicare Compliance for Clinical Laboratories
Case Study 7

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.

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Medicare Compliance for Clinical Laboratories (retired)
Case Study 7

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.

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Microbiology / Serology Question Bank - Review Mode (no CE)
Which of the following statements about Rickettsia is false:View Page

Mycology: Yeasts and Dimorphic Pathogens (retired 2/12/2013)
The colonies shown in the upper image, obtained from a biopsy of an ulcerating skin lesion of the arm, are growing on agar slants of Sabouraud's dextrose agar. The lower image is a photomicrograph of a lactophenol blue mount made from a portion of the colony growing in the left slant. The diagnosis is:View Page
Shown in this photomicrograph is a Gomori methenamine silver stain of a lung biopsy obtained from a patient with X-ray evidence of multi-focal pneumonitis. The yeast most likely to be recovered in culture of this tissue is:View Page

Parasitology Question Bank - Review Mode (no CE)
This suspicious form was recovered from a nodule biopsy. It measures 225 µm in length and lacks a sheath.View Page

Preliminary Identification of the Primary Select Agents of Bioterrorism
Bacillus anthracis

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!

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Francisella tularensis

Clinical specimens where organism may be encountered: Blood Biopsy, skin scraping, or swab Lymph node aspirate Respiratory secretions - oropharyngeal aspirate, sputum, or bronchial washingsGram stain morphology: Very tiny, gram-negative coccobacillus Pale or weak staining Due to the small size, often difficult to see individual cells

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Theoretical and Practical Aspects of Routine H&E Staining
Liver

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.

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