Specimen Collection

Table of Contents

ROUTINE COLLECTION & HANDLING OF BIOPSIES

  1. 1.01: Collection Into 10% Formalin Fixative
  2. 1.02: Handling Biopsy Specimen Containers
  3. 1.03: Filling-In The Pathology Requisition
  4. 1.04: Specimen Transport Bag
  5. 1.05: Submitting Clinical Specimens & Biopsy Specimens Obtained From the Same Patient

SPECIAL COLLECTION & HANDLING OF BIOPSIES

  1. 1.40: Alternative To Collection Into 10% Formalin
  2. 1.41: Lymph Nodes
    1. Sterile Sectioning
  3. 1.42: Immunofluorescence—Zeuss Media
  4. 1.43: Karyotyping
    1. Holding Media
    2. Time-Sensitive Processing
  5. 1.44: Flow Cytometry—EDTA
    1. Holding Media—Sodium Heparin (EDTA—Green Top Test Tube)
    2. Time-Sensitive Processing

FROZEN SECTIONS

  1. 1.50: Frozen Section Scheduling And Notification
  2. 1.51: Collection—Unfixed
  3. 1.52: Pathology Requisition
  4. 1.53: Special Handling

CREUTZFELDT-JAKOB DISEASE

  1. 1.60: OCPMG Does Not Process Cases Suspected Of Having Creutzfeldt-Jakob Disease.

COLLECTION & HANDLING OF NON-GYN CYTOLOGY SPECIMENS

  1. 2.01: Fixation and Submission
    1. Roles of Fixation
    2. Non-Gyn Fluid Specimens
    3. FNA and Other Non-Gyn Specimen Smears
      1. FNA smears: Unfixed (Air-Dried) and Fixed with 95% Alcohol Liquid Fixative
      2. Other Smears: Fix with 95% Alcohol
      3. Ordering 95% Alcohol Fixative Bottles
    4. d.Label Slides Before Collection
  2. 2.02: Handling Cytology Specimen Containers
  3. 2.03: Filling-In The Pathology/Cytology Requisition
  4. 2.04: Specimen Transport Bag
  5. 2.05: Submitting Clinical Specimens & Non-Gyn Cytology Specimens Obtained From The Same Patient

NON-GYN CYTOLOGY SPECIMEN COLLECTION & SUBMISSION

  1. 5.01: BODY FLUIDS FOR CYTOLOGY
    (PLEURAL FLUID—ASCITIC FLUID—Col-DE-SAC FLUID—PERICARDIAL FLUID)

    1. Materials Required
    2. Procedure
  2. 5.02: BRUSHINGS FOR CYTOLOGY
    (BRONCHIAL BRUSHINGS—COLONIC BRUSHINGS—ESOPHAGEAL BRUSHINGS—GASTRIC BRUSHINGS)

    1. Materials Required
    2. Procedure
  3. 5.03: CEREBRAL SPINAL FLUID (“CSF”)
    1. Materials Required
    2. Procedure
  4. 5.04: NIPPLE SECRETION SMEARS (CYTOLOGY)
    1. Materials Required
    2. Procedure
    3. Comments
  5. 5.05: FINE NEEDLE ASPIRATION (CYTOLOGY)
    1. Materials Required
    2. Procedure
  6. 5.06: SPUTUM FOR CYTOLOGY
    1. Materials Required
    2. Procedure
    3. Comments
  7. 5.07: URINE FOR CYTOLOGY
    1. Materials Required
    2. Procedure
    3. Comments
  8. 5.08: WASHINGS FOR CYTOLOGY
    1. Materials Required
    2. Procedure
    3. Comments

ROUTINE COLLECTION & HANDLING OF BIOPSIES (SURGICAL TISSUES FOR PATHOLOGIC DIAGNOSIS)

  1. 1.01: COLLECTION INTO 10% FORMALIN FIXATIVE
    1. Biopsy specimens should be immediately placed into 10% formalin unless a different or additional fixative is specified for a specialized study. Biopsies should always be entered into fixative without delay. The volume of formalin should be 10 to 20 times the volume (size) of the tissue specimen.
    2. Tissue samples from different sites or lesions on the same patient are considered separate specimens and should be submitted in separate containers.
  2. 1.02: HANDLING BIOPSY SPECIMEN CONTAINERS
    1. Secure the container lid tightly to avoid loss of formalin. Formalin may cause irritation if contacted by the skin, and could be fatal if ingested. It is listed as a class III carcinogen, and we will provide you with a material safety data sheet upon request. Always handle all chemicals with care.
    2. Label each container with the patient’s name, date of collection, and biopsy site and identification—for example, “lesion left upper back,” “shave bx right side of nose,” “cervical bx 2 o’clock,” etc.
  3. 1.03: FILLING-IN THE PATHOLOGY REQUISITION
    1. Complete the pathology requisition form including the name of the physician referring the specimen to the laboratory, the patient’s name, date of collection, complete billing information and biopsy identification. An incomplete requisition may require the laboratory to reject and return the specimen unprocessed. A completed requisition must be placed in the appropriate pocket of each specimen transport bag.
    2. Also include on the pathology laboratory requisition all of the following:
      1. Any special interests or requirements for the pathologist, for example: “pneumocystis carinii” or other relevant clinical history;
      2. Special reporting requirements, e.g., “Telephone Report;”
      3. If a re-biopsy of a lesion, please provide date and specimen number of previous biopsy;
      4. Note any recent change of patient’s name;
      5. With endometrial biopsies or curettings, list the age, LMP and all exogenous hormones the patient is or has recently received, including birth control pills and hormone replacement therapy (HRT);
      6. With cervical, endocervical or other gynecological biopsies, always provide dates and specimen numbers of the patient’s abnormal Pap tests; and
      7. If more than one transport bag on the same patient is being submitted, please include a requisition in each bag. Indicate on each requisition that the specimen bag contains only a portion of the specimens being simultaneously submitted: For example, “bag 1 of 2,” or “specimens 3 and 4 are separately submitted.” This will alert laboratory personnel to look for the companion transport bags so that all specimens on the same case are accessioned with the identical case number.
    3. ALWAYS complete a clinical laboratory requisition when submitting cultures, flow cytometry and other clinical tests being requested on part of a biopsy case (include bone marrow cases). The clinical requisition and biopsy part on which clinical studies are requested should be placed into separate transport bags.
  4. 1.04: SPECIMEN TRANSPORT BAG
    1. Place specimen container(s) in one pouch of a plastic “transport bag” and the corresponding requisition in the other pouch. Make sure that the printed side of the requisition is visible through the bag. Remove any protective strip from the top edge of the bag and seal completely for safe transport to the laboratory.
    2. NEVER place specimens from different patients into the same transport bag.
  5. 1.05: SUBMITTING CLINICAL AND BIOPSY SPECIMENS OBTAINED FROM THE SAME PATIENT
    1. Sometimes a swab for culture and sensitivity will be obtained just prior to biopsy. Culture swabs, blood tubes for CBC, etc., and other clinical specimens should be submitted on a clinical requisition and sealed in a separate transport bag.
    2. If culture and tissue examinations are required on the same biopsy sample, be certain to separate part of the sample for the culture BEFORE placing the other part in formalin. The sample for culture should be submitted in a sterile container without formalin. Follow the instructions for submitting cultures provided by your clinical laboratory. However, sterile transport media is generally considered optimum to preserve microorganisms; and sterile saline may be introduced into the specimen container in the absence of transport media. Submitting of tissue for culture without transport media or sterile saline risks loss of viability of any microorganisms present and is not recommended.

SPECIAL COLLECTION & HANDLING OF BIOPSIES(SURGICAL TISSUES FOR PATHOLOGIC DIAGNOSIS)

  1. 1.40: ALTERNATIVE TO COLLECTION INTO 10% FORMALIN
    • IF 10% formalin is not available for fixation of routine surgical tissue, other fixatives such as Bouin’s may be used.
  2. 1.41: SPECIAL FIXATIVES OR PROCEDURES—LYMPH NODES
    1. STERILE SECTIONING.Prior to fixation, if clinically suspicious or unknown for infection, the lymph node should be sterilely sectioned with a small 2mm cube submitted for cultures (routine, AFB, fungal, etc.). An additional portion, 5mm cube, may be sent for genetic karyotyping if indicated, with the remainder submitted for fixation.
    2. LYMPH NODES CLINICALLY SUSPICIOUS FOR LYMPHOMA.Submit fixed in 10% (neutral, buffered) formalin.
  3. 1.42: IMMUNOFLUORESCENCE – ZEUSS MEDIA
    • Tissue sent for immunofluorsescence should preferably be sent in a special holding media such as Zeuss media or immediately sent fresh in saline. This tissue must not be sent in formalin or other fixative. If unavailable, please call OCPMG and make arrangements to get such a media to your office prior to the biopsy.
  4. 1.43: KARYOTYPING.
    1. Genetic karyotyping of tumors requires that the tissue be placed in a holding media. Tissue must be submitted in as sterile a manner as possible to avoid bacterial overgrowth. The nutritional holding media must also be sterile. If unavailable, please call OCPMG and make arrangements to get such a media to your office.
    2. Tissue should be processed within 24 hours, although, karyotyping may be successful on tissue held 48-72 hours.
  5. 1.44: FLOW CYTOMETRY—EDTA
    1. Tissue or fluids for flow cytometry testing should be placed in a holding media prior to testing. This can be a sodium heparin solution or EDTA (green-top test tube), or a holding media as in ¶1.41, above.
    2. Testing should preferably be done within 24 hours although it may be successful on tissue held 48-72 hours.

COLLECTION & HANDLING OF FROZEN SECTION SPECIMENS(SURGICAL TISSUES FOR RAPID DIAGNOSIS)

  1. 1.50: FROZEN SECTION SCHEDULING AND NOTIFICATION.
    1. In general, we will schedule a pathologist to perform frozen section pathology on your specimen provided we are notified at least 24 hours in advance. With 24-hour advance notice, a pathologist is available from 7:30 a.m. to 6:00 PM for an O.R. Consult, Touch Prep or Frozen Section procedure. If less than 24-hours advance notice, we will make a reasonable effort to have a pathologist available.
    2. To schedule a pathologist for an O.R. Consult, Touch Prep or Frozen Section procedure, call OCPMG at (714) 288-4044.
    3. To request a pathologist for an unscheduled O.R. Consult, Touch Prep or Frozen Section procedure, call OCPMG at (714) 288-4044 between the hours of 8:30 a.m. and 4:30 PM. For unscheduled procedures earlier than 8:30 a.m. or after 4:30 PM, call the Pathology Department at St. Joseph Hospital (714) 771-8176 or (714) 633-9111. NOTE: It is not always possible to have a pathologist available to cover an unscheduled procedure. We strongly advise, therefore, that all procedures be scheduled at least 24-hours in advance.
    4. To confirm that a pathologist is on station or currently available before beginning the surgical procedure, or to give notice that the specimen has been removed and is ready for examination, call the following departments:
      OCPMG Pathology Laboratory
      805 W. LaVeta, Suite 104, Orange, CA 92868
      (714) 288-4044

      St. Joseph’s Hospital, Pathology Department
      1100 W. Stewart Dr., Orange, CA 92868
      (714) 771-8176 or 771-8175.

  2. 1.51: COLLECTION—UNFIXED
    1. Place the tissue into a clean, empty container or on a moist towel so that the tissue does not dry out. Tissue removed for frozen section rapid pathologic diagnosis must not be placed into formalin or any other fixative. It must be submitted fresh and moist.
    2. Make sure there is sufficient tissue so that both a frozen section sample and a sample for permanent sectioning and routine examination are possible.
  3. 1.52: PATHOLOGY REQUISITION
    1. Before surgery, complete a pathology requisition form—including the name of the referring physician/surgeon, patient’s name, date of collection, complete biopsy identification (tissue type, site, orientation, etc.), and all billing information.
    2. Also include on the pathology requisition all relevant clinical history and clinical diagnosis.
    3. Include the TELEPHONE NUMBER where you want the frozen section diagnosis to be called.
  4. 1.53: SPECIAL HANDLING
    1. Unless other arrangements are made between the surgeon and the pathologist, Touch Prep specimens are scheduled for examination by the pathologists at OCPMG Pathology Laboratory, 805 W. La Veta Ave., Suite 104, Orange, CA 92868 (714) 288-4044, or at St. Joseph Hospital, 1100 W. Stewart Drive, Pathology Dept., Orange, CA (714) 771-8176. The Touch Prep specimen(s) should be brought to the scheduled location fresh and unfixed (see above).
    2. Specimens for Frozen Section must be delivered to the pathologist at once, fresh and unfixed. Specimens for Frozen Section obtained at Main Street Specialty Surgery Center will be delivered to the Pathology Room at that surgery center; specimens for Frozen Section obtained elsewhere will be delivered as directed by the pathologist.
    3. Frozen section results are routinely called to the surgeon within 20 minutes from the time the specimen is received by the pathologist.

CREUTZFELDT-JAKOB DISEASE

  1. 1.60: THE LABORATORY SPECIFICALLY EXCLUDES PROCESSING OF CASES SUSPECTED OF HAVING CREUTZFELDT-JAKOB DISEASE (“CJD”). As a result of health and safety concerns, any specimen suspected of having CJD will be returned to the submitting office, unprocessed.

COLLECTION & HANDLING OF NON-GYN CYTOLOGY SPECIMENS

  1. 2.01: FIXATION AND SUBMISSION
    1. Rules of Fixation. Cellular degeneration begins immediately upon removal from the body, and can lead to an inconclusive or unsatisfactory test result. However, specimen cellular degeneration can be slowed with refrigeration of an unfixed, fluid specimen or by fixation with an equal volume of 95% alcohol. Refer to non-gyn body fluid or FNA specimen methods in procedures 5.01 – 5.08, below.
    2. Non-Gyn Fluid Specimens For fluid non-gyn specimen samples, cellular degeneration is generally slowed when the cells remain suspended in their own body fluid (e.g., urine, pleural fluid, etc.), and these types of specimens are usually submitted, unfixed, in their original collection bag/container. However, it is best to refrigerate unfixed non-gyn specimens at all times practicable until they are processed at the pathology laboratory, including the time they remain in your office awaiting courier pick-up. To retard cellular degeneration during transport and holding, OCPMG couriers carry cold packs and laboratory staff is instructed to refrigerate unfixed non-gyn specimens until processing begins. (See Procedures 5.01-5.08, below.)
    3. FNA and Other Non-Gyn Specimen Smears
      1. Fine needle aspiration (FNA) specimens are generally expressed onto microscope slides at the point of collection. It is common practice to submit 4 slides unfixed (air-dried) for rapid staining by Diff-Quik method, and 2 slides immersed in 95% alcohol for routine non-gyn cytology staining. (See Procedure 5.05, below.)
      2. Other non-gyn specimen smears may be fixed by immersing in 95% alcohol or by fixing with a commercially prepared cytology spray fixative. (The laboratory no longer supplies spray fixative, and recommends 95% alcohol instead.)
      3. Non-Gyn Specimen Slide Bottles containing 95% alcohol may be ordered from OCPMG Pathology Laboratory by calling the Client Supplies Department at (714) 288-4044.
      CAUTION: Isopropyl Alcohol is flammable and poisonous if ingested. Skin irritant.
    4. Label Slides Before Collection. Using pencil, label the specimen slides with the patient’s name before the procedure begins so that labeling does not delay fixation. Labeling with a pencil is strongly recommended because many inks will dissolve in 95% alcohol or in the staining process at the laboratory.
  2. 2.02: HANDLING CYTOLOGY SPECIMEN CONTAINERS
    1. Secure the container lid tightly to avoid loss of alcohol fixative. Alcohol fixative may cause skin irritation, and could be harmful if swallowed. Always handle all chemicals with care. (If you are using a cytology spray fixative, allow spray fixative to air-dry before placing the slide into a slide holder, otherwise, the slide might stick to the holder and break during removal. The laboratory no longer supplies spray fixative, and recommends 95% alcohol instead.)
    2. Label each container or slide holder with the patient’s name, date of collection, specimen site and identification, for example, “voided urine,” “catheterized urine,” “right breast FNA,” etc.
    3. Non-Gyn Cytology samples from different sites, such as several aspirations of the same breast (lateral upper quadrant; below nipple; medial lower quadrant, etc.) should be submitted in separate containers.
  3. 2.03: FILLING-IN THE PATHOLOGY/CYTOLOGY REQUISITION
    1. Complete the pathology requisition form including the name of the physician referring the specimen to the laboratory, the patient’s name, date of collection, all billing information and specimen identification. A patient’s “label” containing at least all of the above may be affixed to the requisition. An incomplete requisition may require the laboratory to reject and return the specimen unprocessed.
    2. Also include on the pathology laboratory requisition all of the following:
      1. Any special interests or requirements for the pathologist, for example: “Pneumocystis carinii” or other relevant clinical history;
      2. Special reporting requirements, e.g., “Telephone Report;”
      3. Please provide dates and specimen numbers of relevant, previous pathology testing (prior biopsies, pap smears, non-gyns);
      4. Note any recent change of name of the patient; and
      5. If more than one specimen on the same patient is being submitted at the same time, inform the pathologist on the pathology requisition of the other test that was separately submitted.
    3. ALWAYS complete a Clinical Laboratory Requisition (e.g., Unilab / Quest Diagnostics) for cultures and other clinical tests. Do not combine clinical test requests and pathology test requests on the same requisition. The clinical requisition and specimen and the pathology/non-gyn cytology requisition and specimen should be placed into separate transport bags. (See Below.)
  4. 2.04: SPECIMEN TRANSPORT BAG
    1. Place specimen container(s) in one pouch of a plastic “transport bag” and the corresponding requisition in the other pouch. Make sure that the printed side of the requisition is visible through the bag. Remove any protective strip from the top edge of the bag and seal completely for safe transport to the laboratory.
    2. NEVER place specimens from different patients in the same transport bag.
  5. 2.05: CLINICAL AND CYTOLOGY SPECIMENS — SAME PATIENT
    1. If culture and non-gyn cytology examinations are required on the same sample, make certain to separate part of the sample for the culture BEFORE placing the other part in fixative. The sample for culture should be submitted in a sterile container without fixative. Follow the instructions for submitting cultures provided by your clinical laboratory.

NON-GYN SPECIMEN COLLECTION & SUBMISSION BODY FLUIDS FOR CYTOLOGY
(Pleural * Ascitic * Cul-de-Sac * Pericardial)

  1. 5.01-1: MATERIALS NEEDED
    • Specimen collection container, liquid-tight.
    • Refrigerate if practicable (or, may fix with 50% to 95% alcohol).
    • Anticoagulant may be used (if clinically indicated).
    • Pathology (Cytology) requisition form.
    • Specimen transport bag.
  2. 5.01-2:PROCEDURE
    1. Label Container.
      Label collection container with patient’s name, specimen source, name of submitting physician and date of collection.
    2. Requisition.
      Fill-in all pertinent information on the requisition. (See “Collection & Handling of Cytology,” ¶ 2.03, supra.)
    3. Anticoagulant.
      Exudates may be prevented from forming clots by collecting the specimen into a container with anticoagulant, such as Heparin (5 to 10 units per ml of fluid), 3.8% Sodium Citrate (1 ml per 10 ml of fluid), or EDTA (1 mg per 1 ml of fluid). Place anticoagulant in collection container, if indicated, and add specimen to container with gentle swirling motion.
    4. Clots & Particles.
      Include in the container any clots or particles that are present in the specimen and we will try to make a Cell Block.
    5. Fixation.
      Keep specimen refrigerated at all times practicable in the office and during transport to the laboratory. Alternatively, you may fix the specimen by adding an equal volume of 50% to 95% alcohol.
    6. Transport Bag.
      Place specimen container into one pouch and the completed requisition into the other pouch, and seal bag for pick-up.
    7. Cultures.
      Specimens requiring culture must be separately submitted in sterile containers (without fixative), and accompanied by a clinical laboratory requisition.

    Reference: Koss, L., Diagnostic Cytology, Lippincott, 1979.

BRUSHINGS FOR CYTOLOGY
(Bronchial * Colonic * Esophageal * Gastric)

  1. 5.02-1: MATERIALS NEEDED
    • Specimen collection container, liquid-tight.
    • Microscope slides.
    • Spray fixative (optional).
    • 50% or 95% alcohol in a specimen container (to receive slides—or for coagulum and tissue fragments recovered during the procedure).
    • Pathology (Cytology) requisition form.
    • Specimen transport bag.
  2. 5.02-2PROCEDURE
    1. Label Slides And Container.
      Label slides with patient’s name and specimen source (e.g., LUL for “left upper lobe”). Label specimen container with patient’s name, specimen source, name of submitting physician and date of collection.
    2. Requisition.
      Fill-in all pertinent information on the requisition. (See “Collection & Handling of Cytology,” ¶ 2.03, supra.) Indicate any special stains you may want performed. Also, provide relevant patient history, such as: “prior malignancy of _______,” etc.
    3. Specimen.
      Obtain brush sample and gently but rapidly roll brush over the surface of the microscope slide. Immediately spray fix and/or immerse in a liquid-tight specimen container filled with 50% or 95% alcohol. Note: crushed or air-dried brushing smears are of limited diagnostic value.
    4. Coagulum & Tissue Fragments.
      Include in the container any coagulum and tissue fragments that are recovered.
    5. Washings.
      If washings are obtained concurrently with the brushing specimen, add washings to a separate specimen container. Keep refrigerated and transport, cold, to the laboratory if practicable. Alternatively, you may fix the washing specimen by adding an equal volume of 50% or 95% alcohol.
    6. Transport Bag.
      Place specimen container into one pouch and the completed requisition into the other pouch, and seal bag for pick-up.
    7. Cultures.
      Specimens requiring culture must be separately submitted in sterile containers (without fixative), and accompanied by a clinical laboratory requisition.

    Reference: Koss, L., Diagnostic Cytology, Lippincott, 1979.

CEREBRAL SPINAL FLUID (“CSF”) FOR CYTOLOGY

  1. 5.03-1: MATERIALS NEEDED
    • Specimen collection container, liquid-tight. If specimen is to remain in the collecting syringe, you must remove the needle before submitting to the laboratory.
    • Refrigerate if practicable (or, may fix by adding specimen to an equal amount of 50% or 95% alcohol in a liquid-tight specimen container).
    • Pathology (Cytology) requisition form.
    • Specimen transport bag.
  2. 5.03-2: PROCEDURE
    1. Label Container.
      Label specimen container with patient’s name, specimen source, name of submitting physician and date of collection.
    2. Requisition.
      Fill-in all pertinent information on the requisition. (See “Collection & Handling of Cytology,” ¶ 2.03, supra.) Indicate any special stains you may want performed. Also, provide relevant patient history, such as: “prior malignancy of _______,” etc.
    3. Specimen.
      Perform spinal tap using needle size and technique of choice.
    4. Fixation.
      Keep refrigerated and transport, cold, to the laboratory if practicable. Alternatively, you may fix the CSF specimen by adding an equal volume of 50% or 95% alcohol. Note: If there is sufficient cellular material a Cell Block may be prepared by the laboratory.
    5. Transport Bag.
      Place specimen container into one pouch and the completed requisition into the other pouch, and seal bag for pick-up.
    6. Cultures.
      Specimens requiring culture must be separately submitted in sterile containers (without fixative), and accompanied by a clinical laboratory requisition.

    Reference: Koss, L., Diagnostic Cytology, Lippincott, 1979.

NIPPLE SECRETION SMEARS FOR CYTOLOGY

  1. 5.04-1: MATERIALS NEEDED
    • Microscope slides (up to 4 per nipple).
    • Spray fixative (or, may fix by placing slides in 95% alcohol in a liquid-tight specimen container).
    • Physiologic saline and cotton swab.
    • Pathology (Cytology) requisition form.
    • Specimen transport bag.
  2. 5.04-2: PROCEDURE
    1. Label Slides (And Container).
      Label slides with the patient’s name and specimen site. Label specimen container, if any, with patient’s name, specimen source, physician and date of collection.
    2. Requisition.
      Fill-in all pertinent information on the requisition. (See “Collection & Handling of Cytology,” ¶ 2.03, supra.) Indicate any special stains you may want performed. Also, provide relevant patient history, such as: “prior malignancy of _______,” etc.
    3. Prepare Nipple Area (No Erosion or Ulceration).
      If there is no nipple erosion or ulceration, gently “strip” the area of the breast below the nipple and areola with a motion from beneath the areola towards the nipple surface. Do not massage the entire breast. The stripping motion will propel accumulated secretions within the ampullae of the larger excretory ducts.
    4. Collect Fluid Specimen.
      With the appearance of fluid on the nipple surface, touch a slide to the drop of fluid and draw the slide quickly across the nipple: Immediately apply spray fixative. Repeat the process to collect additional secretion onto more slides.
    5. “Touch Prep” If Erosion or Ulceration Present.
      If there is nipple erosion or ulceration, touch a slide to this area three times, with a different part of the slide in contact each time. This is conveniently done, starting with a contact position close to the hand holding the slide and then moving the application are of the slide further from your hand with the next two samplings. Apply spray fixative immediately.
    6. Following “Touch Prep.”
      After performing a touch preparation (see “e” above) of the ulcerated area, try to express fluid (see “d” above) and prepare slides if any fluid is obtained. If no fluid can be expressed, a swab may be dipped in saline and gently rolled and rotated on the ulcerated surface, then applied to a glass slide and immediately spray fixed.
    7. Transport Bag.
      Place dried specimen slides into cardboard holder and place into one pouch of a transport bag, and place the completed requisition into the other pouch, and seal bag for pick-up.
    8. Cultures.
      Specimens requiring culture must be separately submitted in sterile containers (without fixative), and accompanied by a clinical laboratory requisition.
  3. COMMENTS:
    • WE CANNOT OVER-EMPHASIZE HOW QUICKLY CELLS IN NIPPLE SECRETIONS DEGENERATE. THIS OCCURS EVEN THOUGH THE SAMPLE APPEARS MOIST. PRESENCE OF BLOOD DOES NOT PROTECT THE CELLS FROM DEGENERATION. ONLY IMMEDIATE FIXATION PROTECTS THE CELLS.
    • 95% alcohol may be used as the fixative with or without spray fixative. If alcohol is used, the slides should be transported to the laboratory totally submerged in the alcohol. Liquid-tight containers must be used during transport.
    • Specimen identification should distinguish touch preparations (“TP”) from slides containing expressed fluid.
    • Presence of a breast mass, history of carcinoma or breast biopsies with proliferative ductal changes should be noted on the requisition.

    Reference: Naib, Exfoliative Cytopathology, 3rd ed., 1985.

FINE NEEDLE ASPIRATION (CYTOLOGY)

  1. 5.05-1:MATERIALS NEEDED
    • Microscope slides and transport holders.
    • 95% alcohol in a liquid-tight specimen container.
    • Pathology (Cytology) requisition form and transport bag.
  2. 5.05-2:PROCEDURE
    1. Label Slides (And Container).
      Label slides with the patient’s name and specimen site. Label specimen container, if any, with patient’s name, specimen source, physician and date of collection.
    2. Requisition.
      Fill-in all pertinent information on the requisition. (See “Collection & Handling of Cytology,” ¶ 2.03, supra.) and provide relevant patient history, such as: “prior malignancy of _______,” etc.
    3. Aspiration .
      Perform FNA using needle size and technique of choice.
    4. Prepare Wet-Fixed Smears For Pap Stain.
      Drop a small amount of aspirated material on a microscope slide and do a touch-prep (imprint smear). Immediately immerse slide into 95% alcohol. Repeat as needed.
    5. Prepare Air-Dried Smears.
      Proceed to do 2 to 4 air-dried smears by placing a small drop of material on the slide and spreading it in the manner of a blood smear. Allow these smears to air-dry and submit for Diff-Quick stain (rapid cytopathologic evaluation).
    6. Remaining Aspiration Material.”
      Expel remaining specimen material into a specimen container with 95% alcohol for additional cytopreparation and cell block when appropriate.
    7. Transport Bag.
      Tightly twist lids of specimen containers, place dried specimen slides into cardboard holder and place containers and holders into one pouch of a transport bag, and place the completed requisition into the other pouch, and seal bag for pick-up.
    8. Core Biopsy.
      If a core biopsy is obtained, submit separately as routine biopsy. (See “Collection & Handling of Biopsies,” supra.)

    Reference: Koss, L., DIAGNOSTIC CYTOLOGY, Lippincott, 1992

SPUTUM FOR CYTOLOGY

  1. 5.06-1: MATERIALS NEEDED
    • Liquid-tight specimen container, with 50% to 95% alcohol.
    • Refrigeration required if specimen is submitted unfixed.
    • Pathology (Cytology) requisition form and transport bag.
  2. 5.06-2: PROCEDURE
    1. Label Container.
      Label specimen container with patient’s name, specimen source, physician and date of collection.
    2. Requisition.
      Fill-in all pertinent information on the requisition. (See “Collection & Handling of Cytology,” ¶ 2.03, supra.) Indicate any special stains you may want performed. Also, provide relevant patient history, such as: “prior malignancy of _______,” etc.
    3. Rinse Mouth.
      Have patient rinse mouth.
    4. Breathe Deeply.
      Instruct patient to breathe deeply for 3 minutes.
    5. Produce Sputum.
      Instruct patient to cough deeply, from the diaphragm, with effort to expectorate material into the collection container. At short intervals, repeat steps “d” and “e” three more times and collect all coughed-up material into the same specimen container.
    6. Fixation.
      Add equal volume of 50% or 95% alcohol to the specimen container. Alternatively, submit unfixed if specimen is transported to the laboratory immediately, or refrigerate if unfixed specimen will be delivered to the laboratory within 24 hours.
    7. Transport Bag.
      Tightly twist lid of specimen container and place into one pouch of a transport bag. Place the completed requisition into the other pouch, and seal bag for pick-up and transport to pathology.
  3. COMMENTS:
    1. Deep cough specimens are necessary to provide information regarding the lower respiratory tract.
    2. A series of three sputa collected on three consecutive days, preferably first thing in the morning with hard, productive coughing is recommended. Morning collections take advantage of the accumulation of secretions during the night. If there is difficulty in producing a specimen, collection may be facilitated by the moisture and steam of a preceding long, hot shower.
    3. For patients unable to produce sputum with repeated attempts, consider aerosol induced coughing and specimen collection.
    4. Post-bronchoscopy sputa may be productive of diagnostic information even with negative bronchial washings, brushings and biopsies.
    5. Specimens consisting of saliva or nasopharyngeal drainage will be reported as lacking bronchial columnar cells, metaplastic cells of bronchial origin and pigmented histiocytes. These specimens are inadequate for lesions of the lower respiratory tract and should not be considered as true negative studies.
    6. With sputum samples positive for malignant cells, a primary of the head and neck region should be considered as well as malignancies of the lungs. Up to 10% of positive sputum may reflect malignancies of the head and neck.
    7. Collect the specimen into fixative, or add it immediately after collection to minimize cellular degeneration and artifact which may be caused by the pH, protein content, enzymatic activity and bacterial content of the specimen.
    8. Sputum with a high mucous content may be preserved for 12 to 24 hours if refrigerated. Refrigeration slows the bacterial growth that causes cellular damage and the breakdown of mucus. Mucus protects the cells against rapid degeneration.
    9. Specimens requiring culture must be separately submitted in sterile containers without fixative. A separate requisition form is required with specification of the cultural studies requested, as per the clinical laboratory.
    10. Specify the need for asbestos body examination. Studies will include examination of Pap stained and special stain(s) for ferruginous bodies.
    11. Specify the need for identification of Pneumocystis carinii.

    Reference:Koss, L., DIAGNOSTIC CYTOLOGY, Lippincott, 1979.

URINE FOR CYTOLOGY

  1. 5.07-1: MATERIALS NEEDED
    • Liquid-tight specimen container, with 50% to 95% alcohol.
    • Refrigeration required if specimen is submitted unfixed.
    • Pathology (Cytology) requisition form and transport bag.
  2. 5.07-2: PROCEDURE
    1. Label Container.
      Label specimen container with patient’s name, specimen source, physician and date of collection.
    2. Requisition.
      Fill-in all pertinent information on the requisition. (See “Collection & Handling of Cytology,” ¶ 2.03, supra.) Indicate any special stains you may want performed. Also, provide relevant patient history, such as: “prior malignancy of _______,” etc.
    3. Collect Specimen.
      Introduce specimen into collection container. (See “Comments,” below.)
    4. Fixation.
      Add equal volume of 50% or 95% alcohol to the specimen container. Alternatively, submit unfixed if specimen is transported to the laboratory immediately, or refrigerate if unfixed specimen will be delivered to the laboratory within 24 hours.
    5. Transport Bag.
      Tightly twist lid of specimen container and place into one pouch of a transport bag. Place the completed requisition into the other pouch, and seal bag for pick-up and transport to pathology.
  3. COMMENTS:
    1. Urine should be identified as to type of sample, e.g., voided, catheterized, right or left ureteral, bladder irrigation fluid.
    2. Voided urine is preferably a mid-stream urine.
    3. See Collection Techniques—following, below.
    • Collection Technique #1: For detection of cancer of bladder, ureters or kidneys.
      Consider use of Collection Technique #2 if a serious problem is present with residual urine, severe vaginitis or urethritis, particularly if prior specimens have been unsatisfactory due to cellular degeneration or heavy contamination by inflammatory material.

      1. Have patient drink one glass of water every 15 minutes for 2-3 hours.
      2. At end of 2-3 hours, have patient void and discard urine.
      3. One hour later have patient void and collect specimen.
      4. Add equal volume of 50% or 95% alcohol.
    • Collection Technique #2: For detection of cancer of bladder, ureters or kidneys.
      For use with serious problems of residual urine, severe vaginitis, urethritis or employment of instgruments with investigation of bladder.

      1. Catheterize patient, collect all urine in bladder and discard.
      2. Have patient drink one glass of water every 15 minutes for 2-3 hours.
      3. Collect catheterized urine and add equal volume of 50% or 95% alcohol.

      First morning urines are NOT recommended due to cellular degeneration with longer duration times in the bladder.

    References:Koss, L., Diagnostic Cytology, Lippincott, 1979.
    Naib, Exfoliative Cytopathology, 1970.

WASHINGS FOR CYTOLOGY
(BRONCHIAL * COLONIC * ESOPHAGEAL * GASTRIC)

  1. 5.08-1: MATERIALS NEEDED
    • Liquid-tight specimen container, with 50% to 95% alcohol.
    • Refrigeration required if specimen is submitted unfixed.
    • Microscope slides (if brushings obtained concurrently).
    • Spray Fixative (if brushings obtained concurrently).
    • Pathology (Cytology) requisition form and transport bag.
  2. 5.08-2: PROCEDURE
    1. Label Container.
      Label specimen container with patient’s name, specimen source, physician and date of collection. Any brushing slides must be labeled with patient’s name and specific source—e.g., LUL (left upper lobe), etc.
    2. Requisition.
      Fill-in all pertinent information on the requisition. (See “Collection & Handling of Cytology,” ¶ 2.03, supra.) Indicate any special stains you may want performed (e.g., Pneumocystis carinii). Also, provide relevant patient history, such as: “prior malignancy of _______,” etc.
    3. Collect Specimen.
      Introduce washings into specimen container(s) and label per above with precise identification of origin, e.g., RUB (Right Upper Bronchus), LUB, etc. (See “Comments,” below.)
    4. Fixation.
      Add equal volume of 50% or 95% alcohol to the specimen container. Alternatively, submit unfixed if specimen is transported to the laboratory immediately, or refrigerate if unfixed specimen will be delivered to the laboratory within 24 hours.
    5. Concurrent Brushings.
      If brushings obtained concurrently, rotate brush gently over slide to apply material and immediately spray fix. Provide precise identification of origin. (See “Brushings For Cytology, Sec. 5.02, supra.)
    6. Transport Bag.
      Tightly twist lid of specimen container and place it and any brushing slides (in holders) into one pouch of a transport bag. Place the completed requisition into the other pouch, and seal bag for pick-up and transport to pathology.
  3. COMMENTS:
    1. Specimens requiring culture must be separately submitted in sterile containers without fixative. Submit specimens for culture with a separate requisition per clinical laboratory.

    Reference:Koss, L., Diagnostic Cytology, Lippincott, 1979.