LECTURE # 6
INTRODUCTION TO SPECIMEN GROSSING Rationale: This lecture will help the student understand the different types of tissue received in the majority of histopathology laboratories.
Objective: Once completed this lecture, the student should be able to: a)
Understand the distinction method for grossing specimen received.
Will have a basic understanding of handling Cytology specimens.
Explain the source of the specimens that are received in the histology laboratory.
Will be able to classify all the specimens received in the laboratory.
Discuss the importance of tracking and labeling pathology material such as blocks, slides, reports, and log books.
GROSSING Is the process referred to as grossing a specimen, where tissue specimens taken from routine surgical cases, autopsies, or other scientific investigations are examined, described and trimmed to proper size.
A. What testing is available in the Histology laboratory? 1.
Is considered of a dictation and report describing the physical appearance of the specimen received. This process may vary from hospital to hospital and may be regulated by the medical staff. There are various situations that can lead to a gross only description, but in reality there are two main situations where it is considered a gross only: A. SPECIMEN THAT CANNOT BE PROCESSED 1. Foreign bodies such as: a. Hardware(screws, pins, plates, bone cement) b. Bullets & Breast implants => documentation required since cases of litigations. c. Kidney stones => These are sent out for crystal analysis. d. Dense bone => It is very difficult to process. e. Teeth => It is very difficult to process. B. SPECIMEN THAT NORMALLY “GROSS ONLY” 1. Soft Tissue a. Bony osscicles b. Debridment tissue c. Ear cartilage d. Fat e. Foreskin f. Hernia and hydrocele sacs g. Meninges and meningoceles h. Nasal septa i. Optic lenses j. Placenta from normal deliveries k. Scar tissue l. Semilunar cartilage from knees m. Tendon segment n. Tonsil and adenoids (from patients under 18y/o) o. Vaginal wall fragments from plastic repairs p. Varioceles q. Vein stripping These specimens are examined and described. If any of the areas are suspicious, a section is taken for processing. Results are normally available within 24 hours. 2.
“Gross and Microscopic”
This process will generate a physical description of the specimen received as well as a report of the microscopic findings. The result reported are obtained from the dictation of paraffin blocks that have been processed with dissected tissue during the gross examination. A tissue can generate as little as 1 block to as many as needed. Part of the gross and microscopic finding relies on the hematoxylin and eosin (H&E) stain. The chemical reaction will stain the nuclei blue and the cytoplasm pink. Generally the turn around
time (TAT) is 24 hours, unless decalcification or special stains are ordered, this will prolong the final report. 3.
“Histochemical Stains (Special Stains)”
Special stains are the stains that are used to visualize specific tissue and cellular structures. These are dyes that bind to the cellular components either physically or by chemical bonds. There are several stains that we will discuss in later lectures, these stains are important in order to demonstrate the following properties in tissue: a. b. c. d. e.
Microorganisms Nerves Pigments and Minerals Carbohydrates & Amyloids Connective and Muscle tissue
Normally special stains can be delivered to the pathologist within 24 hours, in some occasions depending on the special stain requested the TAT could be a little longer.
“Immmunohistochemical Stains (IHC)”
IHC stains are used to employ an antibody and make it attach to a specific antigen through a chemical reaction. This reaction is then visualized with a specific chromogen which will allow the viewer to identify the area of interest. These stain may be used in conjunction with special stains to verified and coincide with a specific diagnosis. The common IHC available are: S-100……………………………………….Melanoma Diagnosis HMB-45……………………………………Melanoma diagnosis (More specific than S-100) PSA………………………………………....Prostate Specific Antigen Keratin……………………………………...Skin disease diagnosis CEA………………………………………...Colon cancer diagnosis ER/PR (Estrogen/Progesterone receptors)….Breast Markers These procedures need to be followed accordingly to the establish protocol, a small variation can ruin the whole staining as well as a wrong diagnosis. The TAT is approximately 4 to5 hours. 5.
Normally this procedure is done with frozen sections of skin and kidney. The process is similar to IHC, but it involves the use of a fluorochrome and a fluorescent scope for visualization. Following are the most common fluorochromes used in the laboratory. Ex: IgG, IgM, IgA, Kappa, Lambda, C3, C1q, Fibrin & Albumin The TAT can be faster than the normal IHC staining. 6. “Frozen Sections (FS)” Frozen sections reports include the gross description as well as the findings from freezing a fresh tissue. This is considered a stat process. The patient is still in the OR while this process is taking place to determine the nature of the disease and if the surgery should continue according to the
findings from the frozen section. This process normally takes about 15 to 20 minutes if it’s a simple dissection. 7.
“Electron Microscopy (EM)”
Electron microscopy examination of tissue requires a special handling protocol. Specimen are described, processed and embedded in plastic rather than paraffin. Ultra-thin sections are obtained while working under a microscope. This process once completed will allow the structures of the specimen to be viewed at higher magnification that are not visible on a light microscope. 8.
An autopsy request includes a physical description of the deceased, weight and description of the individual organs, and microscopic findings for each organ or organs discovered abnormal.
B. What testing is available in the Cytology laboratory? 1.
This is the test that everyone known by as “Pap Smear”. It name comes from the Papanicolau test for cancer cells, developed in the 1940’s. Smears are made from cells collected from the cervix of the uterus, stained, and then examined microscopically for evidence of any abnormal cells.
The diagnosis is reported as: CLASS I…………………………………….………….benign CLASS II………………………………………………suspicious CLASS III…………………………………………..…very suspicious CLASS IV……………………………………………..malignant
“Non-Gynecological Cytology” a. Body Fluids 1. Sputum 2. Urine 3. Thoracentesis fluid 4. Synovial fluid 5. Spinal fluid
These are example of fluids that are collected and screened for malignancy. The techniques employed for the screening of these collected cells from the fluids are: 1. Millipore filter – used for fluids with sparse cellularity 2. Cytospin Preparation – a monolayer of cells are spun onto a slide by centrifugation.
3. Smear – diagnosis is made based on staining and screening of a slide made from cellular material of supernatant of centrifuged fluids. 4. Cell Block – cellular material is processed and paraffin embedded. Diagnosis is made from an H&E slide. b. Brushings – are smears made from cells collected as a scraping procedure is being performed. c. Fine Needle Aspiration - A fine needle aspiration samples a site to help determine if it is cancer or not. In the procedure, a small needle is passed through the skin into the thyroid nodule, and suction is applied to the syringe as the needle is moved up and down within the nodule. This aspiration process draws a sample of cells and other material up through the needle into the syringe. The needle is then removed and the sample is placed on a glass slide where it can be stained and examined under a microscope. d. Smears – they are prepared to assist in the diagnosis of many different disorders. Nipple discharge and seminal fluids are examples of smears that are prepared by the physician, and should be received by the lab in fixative, sprayed with a fixative, or air-dried if indicated.
C. Specimen Procurement Tissue specimens received in the histology laboratory come from a variety of places: 1. Hospital Operation Room (inpatient and outpatient) 2. Morgue (autopsy) 3. External Sources (prepared material sent for additional diagnostic assistance) 4. Private Doctor's Office a. A busy dermatologist can remove 25 biopsies in one day of office surgery b. Family physicians will occasionally remove a skin lesion for diagnosis
D. Types of Specimen Submitted to the Histology Laboratory 1. “FIXED TISSUE” The following are examples of fixed tissue sent to the histology laboratory in the appropriate fixative solution: a. Biopsy Biopsies are small sections of different organs removed to determine the presence of pathology possibly affecting more of the organ. Types of biopsies are: 1. Needle…….Threadlike biopsy; prostate most common 2. Punch……...Used to secure skin and bone marrow sections 3. Shave……...Used generally for skin section 4. Ellipse….….Skin biopsy oriented to the patient by use of ink, diagram. sutures, or actual notches; surgical margins cannot be identified without orientation 5. Scrapings……Endocervical, uterine, and prostate (chips) submitted for diagnosis this way 6. Cell block……Generated from bone marrow or cytology specimen b. Organ Removal 1. Uterus 2. Breast 3.Prostate 4.Gall bladder 5.Inflamed appendix 6. Lymph node c. Radical Resection (Whole organ plus associated or nearby lymph nodes) 1. Breast 2. Prostate 3. Neck d. Foreign Bodies All of these specimens are sent t9 the histology laboratory for documentation and diagnostic purposes. They may be used as evidence in criminal or liability cases. e. Eyes Eye processing is an exact science, not usually found handled by the routine histology laboratory. f. Kidney Stones These specimens are sent for crystallographic analysis. Information from this analysis is used by the physician to help the patient from forming additional stones. g. Soft Tissue for Documentation Refer to the list of tissues under the previous section of "Gross Only". Remember: these specimens are thoroughly examined for any anomaly.
h. Fetuses Up until 20 weeks gestation, a fetus is considered surgical tissue. After 20 weeks gestation, the fetus must be buried (sometimes after an autopsy is performed) rather than disposed of as other surgical material (incineration). (Note: although it is not acceptable to refuse to work with HIV infected material in the medical setting (laboratory), it is legal to refuse to work with abortion tissue due to stated religious beliefs concerning the abortion process) Gestational age of a fetus is determined by a measurement of the distance from the crown to the rump. i. Autopsy Tissue removed during an autopsy is submitted to histology for processing. 2. â€œFresh Tissueâ€? a. Frozen Sections Tissue for frozen sections must be fresh. A specimen that has been accidentally fixed for a few short minutes may be rescued, thoroughly washed, and processed as fresh tissue, in most cases. Frozen sections are performed to demonstrate: 1. Diagnosis 2. Staging of a malignancy 3. Temporal arteries 4. Successful removal of parathyroid gland tissue 5. Special staining procedures b. Electron Microscopy Specimens that are to be scanned by an electron microscope must be specially processed. The tissue must be placed in fixative within fifteen minutes after removal from the patient. Autolysis quickly destroys the organelles of the cell necessary for the successful diagnosis. Tissue for EM should be minced into fragments no larger than 1 mm. Follow lab protocol for choice of fixative. Often EM testing is ordered on tissue once a diagnosis is concluded by frozen section. What consideration is important for these specimen.? c. Kidney Biopsy These specimens are usually removed in the radiology department. They are sent to the lab for verification that the biopsy has included enough glomeruli for diagnosis. The radiologist will await your call before proceeding. It will sometimes take up to three passes to collect a good specimen. A kidney biopsy is usually processed by partitioning the specimen into three parts: one for light microscopy; one for electron microscopy; and one for immunofluorescence studies. These tests require specific fixatives that will be discussed in later lectures. d. Heart Muscle Biopsy This is very specialized, critical in nature. and requires strict adherence to established protocol. e. Nerve Biopsy Handling of these biopsy specimens is also very specialized and requires strict adherence to established protocol. f. Specimens for Immunohistochemical Procedures Tests for such things as estrogen progesterone hormone receptor levels may be performed on paraffin embedded tissue, but specificity is more reliable on fresh tissue. Small samples of tissue are removed from breast biopsies that have been diagnosed by frozen section.
g. Muscle Biopsy Muscle biopsies are removed from the patient and sent to the lab clamped in a pair of forceps, or sutured to a tongue depressor on both ends. The muscle must be measured, dissected, frozen, and fixed according to individual procedure. (Note: liquid nitrogen and isopentane are recommended cryogenics for freezing tissue specimens. Ice crystals easily form if the freezing process i... too slow.) h. Research Projects Protocols for current research projects should be posted. Protocols are very specific and should be followed.
i. Amputations An amputated limb will be sent to the lab fresh for dissection. Obvious reasons dictate this practice. Specimens should be refrigerated until dissection and then again until disposal. j. Routine Specimens Individual laboratory procedure may dictate that specimens be sent to the lab in a fresh state. Always... question yourself: (1) Why is the specimen not in fixative? (2) What kind of specimen this? and (3) I... there a protocol that needs to be followed? BEFORE you submerge the fresh tissue in fixative!
E. Tracking and Labeling Tracking and labeling procedures vary from lab to lab. Most modern laboratories use an accessioning system that is color-coded. However, there are many different effective methods in use. and it is the responsibility of the trainee/new employee to thoroughly investigate the system in place at his/her facility. 1. â€œAccessioningâ€? Specimens are accessioned upon receipt in the laboratory. Accession numbers are usually consecutive counts that continue for a year. For example. 97-512 would indicate case number 512 of the year 1997. The accession number is usually assigned an acronym to indicate which record book has been used to log in the specimen. SP is used commonly to denote surgical pathology, while CY is used for cytology specimens. Accession Number
The entire accession number should be written on the specimen container (not on the lid) and specimen requisition, not covering patient identification, and not covering the formaldehyde warning label! The accession number will now readily follow the specimen and guarantees confidentiality.
2. Embedding Cassettes Embedding cassettes are usually plastic, or a combination of plastic with a metal lid. The accession number should be written on the appropriate side of the cassette. Individual procedure will dictate what information goes where. A 1-2-3 or A-B-C designation may be added to the accession number at this point to indicate orientation for the pathology report. Example: SP-97-512A could indicate a section of cervix that has been dissected by the pathologist during the gross of the uterus. SP-97-512B could indicate a section of endometrium that has been dissected by the pathologist during the same grossing of the uterus. Embedding molds of differing colors may also be used to relay information. For example, blue may be used to indicate biopsy, orange may be used for private dermatologist work, and white may be used to indicate a specimen from autopsy.
4. Reports Reports are typed, signed and reported with the patient information, pertinent clinical data, accession number, gross and diagnosis.
5. Storage Reports, slides, blocks and log books are all filed by accession number. Attempts to recover any filed pathology material will depend greatly on knowing the accession number. Consequently, there must be some system in place to locate accession numbers of reports, particularly those that have been in storage before the lab became computerized. Federal and state regulations determine the length of time that pathology material must be stored. Typically in Florida, tissue slides are kept for 10 years and paraffin blocks are kept for 10 years.